Lipoprotein-regulating medicaments

ABSTRACT

Methods and pharmaceutical compositions useful for modulating lipoprotein levels in vivo. The invention stems from the discovery that activity of the Lipolysis Stimulated Receptor (LSR) can be inhibited or enhanced by exogenous agents, including polypeptides.

CROSS-REFERENCE TO RELATED APPLICATION(S)

The present application is a divisional of U.S. application Ser. No.09/485,316, filed Feb. 4, 2000, and now U.S. Pat. No. 6,344,441, issuingon Feb. 5, 2002, which is a National Stage Application of InternationalApplication Number PCT/IB98/01256, tiled Aug. 6, 1998, which is herebyincorporated by reference herein in its entirety, including any figures,tables, nucleic acid sequences, amino acid sequences, and drawings.

FIELD OF THE INVENTION

The present invention relates to medicaments that are useful formodulating lipoprotein levels in vivo. More particularly, the inventionrelates to medicaments that modify the activity of the LipolysisStimulated Receptor (LSR) and that can be used to influence thepartitioning of dietary lipids between the liver and peripheral tissues,including adipose tissue.

BACKGROUND OF THE INVENTION

Obesity is a public health problem which is both serious and widespread.One-third of the population in industrialized countries has an excessweight of at least 20% relative to the ideal weight. The phenomenoncontinues to worsen, particularly in regions of the globe whereeconomies are modernizing. In the United States, the number of obesepeople has escalated from 25% at the end of the 70s to 33% at thebeginning of the 90s.

Obesity considerably increases the risk of developing cardiovascular ormetabolic diseases. It is estimated that if the entire population had anideal weight, the risk of coronary insufficiency would decrease by 25%and that of cardiac insufficiency and of cerebral vascular accidents by35%. Coronary insufficiency, atheromatous disease and cardiacinsufficiency are at the forefront of the cardiovascular complicationsinduced by obesity. For an excess weight greater than 30%, the incidenceof coronary diseases is doubled in subjects under 50 years. Studiescarried out for other diseases are equally eloquent. For an excessweight of 20%, the risk of high blood pressure is doubled. For an excessweight of 30%, the risk of developing a non-insulin-dependent diabetesis tripled. That of hyperlipidemias is multiplied six fold.

The list of diseases having onsets promoted by obesity is long:hyperuricemia (11.4% in obese subjects, against 3.4% in the generalpopulation), digestive pathologies, abnormalities in hepatic functions,and even certain cancers.

Whether the physiological changes in obesity are characterized by anincrease in the number of adipose cells, or by an increase in thequantity of triglycerides stored in each adipose cell, or by both, thisexcess weight results mainly from an imbalance between the quantities ofcalories consumed and those of the calories used by the body. Studies onthe causes of this imbalance have been in several directions. Some havefocused on studying the mechanism of absorption of foods, and thereforethe molecules which control food intake and the feeling of satiety.Other studies have characterized the pathways through which the bodyuses its calories.

The treatments for obesity which have been proposed are of four types.Food restriction is the most frequently used. The obese individuals areadvised to change their dietary habits so as to consume fewer calories.This type of treatment is effective in the short-term. However, therecidivation rate is very high. The increase in calorie use throughphysical exercise is also proposed. This treatment is ineffective whenapplied alone, but it improves, however, weight-loss in subjects on alow-calorie diet. Gastrointestinal surgery, which reduces the absorptionof the calories ingested, is effective but has been virtually abandonedbecause of the side effects which it causes. The medicinal approach useseither the anorexigenic action of molecules involved at the level of thecentral nervous system, or the effect of molecules which increase energyuse by increasing the production of heat. The prototypes of this type ofmolecule are the thyroid hormones which uncouple oxidativephosphorylations of the mitochondrial respiratory chain. The sideeffects and the toxicity of this type of treatment make their usedangerous. An approach which aims to reduce the absorption of dietarylipids by sequestering them in the lumen of the digestive tube is alsoin place. However, it induces physiological imbalances which aredifficult to tolerate: deficiency in the absorption of fat-solublevitamins, flatulence and steatorrhoea. Whatever the envisagedtherapeutic approach, the treatments of obesity are all characterized byan extremely high recidivation rate.

The molecular mechanisms responsible for obesity in man are complex andinvolve genetic and environmental factors. Because of the low efficiencyof the treatments known up until now, it is urgent to define the geneticmechanisms which determine obesity, so as to be able to develop bettertargeted medicaments.

More than 20 genes have been studied as possible candidates, eitherbecause they have been implicated in diseases of which obesity is one ofthe clinical manifestations, or because they are homologues of genesinvolved in obesity in animal models. Situated in the 7q31 chromosomalregion, the OB gene is one of the most widely studied. Its product,leptin, is involved in the mechanisms of satiety. Leptin is a plasmaprotein of 16 kDa produced by the adipocytes under the action of variousstimuli. Obese mice of the ob/ob type exhibit a deficiency in the leptingene; this protein is undetectable in the plasma of these animals. Theadministration of leptin obtained by genetic engineering to ob/ob micecorrects their relative hyperphagia and allows normalization of theirweight. This anorexigenic effect of leptin calls into play a receptor ofthe central nervous system: the ob receptor which belongs to the familyof class 1 cytokine receptors. The ob receptor is deficient in obesemice of the db/db strain. The administration of leptin to these mice hasno effect on their food intake and does not allow substantial reductionin their weight. The mechanisms by which the ob receptors transmit thesignal for satiety are not precisely known. It is possible thatneuropeptide Y is involved in this signalling pathway. It is importantto specify at this stage that the ob receptors are not the onlyregulators of appetite. The Melanocortin 4 receptor is also involvedsince mice made deficient in this receptor are obese (Gura, Science275:751 (1997)).

The discovery of leptin and the characterization of the leptin receptorat the level of the central nervous system have opened a new route forthe search for medicaments against obesity. This model however, rapidlyproved disappointing. Indeed, with only one exception (Montague et al.,Nature 387:903 (1997)), the genes encoding leptin or its ob receptorhave proved to be normal in obese human subjects. Furthermore andparadoxically, the plasma concentrations of leptin, the satiety hormone,are abnormally high in most obese human subjects.

Clearly there remains a need for novel medicaments that are useful forreducing body weight in humans. Such a pharmaceutical compositionadvantageously would help to control obesity and thereby alleviate manyof the cardiovascular consequences associated with this condition.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to an agent which influencesthe partitioning of dietary lipids between the liver and peripheraltissues for use as a medicament. In one embodiment, this agent can beused for treating a condition in which it is desirable to increase thepartitioning of dietary lipids to the liver, reducing food intake inobese individuals, reducing the levels of free fatty acids in obeseindividuals, decreasing the body weight of obese individuals, ortreating an obesity related condition selected from the group consistingof obesity-related atherosclerosis, obesity-related insulin resistance,obesity-related hypertension, microangiopathic lesions resulting fromobesity-related Type II diabetes, ocular lesions caused bymicroangiopathy in obese individuals with Type II diabetes, and renallesions caused by microangiopathy in obese individuals with Type IIdiabetes. According to another embodiment of the invention, the agentwhich influences the partitioning of dietary lipids between the liverand peripheral tissues is any one of: AdipoQ analogues, AdipoQ homologs,AdipoQ derivatives or fragments of any of the preceding agents. In yetanother embodiment the agent includes an LSR antagonist or an LSRagonist.

Another aspect of the invention relates to a polypeptide that includes aconsensus sequence selected from the group consisting of SEQ ID NO:1 andSEQ ID NO:2 for use as a medicament.

Yet another aspect of the invention relates to a polypeptide comprisingan amino acid sequence which alternatively may have at least 25%homology to one of the sequences of SEQ ID NOs.: 7-14, at least 50%homology to one of the sequences of SEQ ID NOs.: 7-14 or at least 80%homology to one of the sequences of SEQ ID NOs.: 7-14 for use as amedicament.

Still yet another aspect of the invention relates to a C1q polypeptide,derivative, homologue or a fragment of any of the preceding compoundsfor use as a medicament.

A further aspect of the invention relates to an AdipoQ polypeptide or aderivative or homologue thereof or a fragment thereof for use as amedicament.

Another aspect of the invention relates to an Adipose Most Abundant Genetranscipt 1 (ApM1) polypeptide or a derivative, homologue or a fragmentof any of the preceding compounds for use as a medicament.

Still another aspect of the invention relates to the use of a compoundthat influences the partitioning of dietary lipids between the liver andperipheral tissues in the manufacture of a medicament for treating acondition in which the partitioning of dietary lipids to the liver isabnormal or higher than is desirable. In one embodiment, this medicamentcan be used for reducing food intake in obese individuals, reducing thelevels of free fatty acids in obese individuals, decreasing the bodyweight of obese individuals, or treating an obesity related conditionselected from the group consisting of obesity-related atherosclerosis,obesity-related insulin resistance, obesity-related hypertension,microangiopathic lesions resulting from obesity-related Type IIdiabetes, ocular lesions caused by microangiopathy in obese individualswith Type II diabetes, and renal lesions caused by microangiopathy inobese individuals with Type II diabetes. According to a differentembodiment, the compound is one that is selected is any of: AdipoQanalogues, AdipoQ homologs, AdipoQ derivatives, and fragments of any ofthe preceding agents. According to yet a different embodiment thecompound is an agonist or antagonist of the Lipolysis StimulatedReceptor. According to still another embodiment the compound can be anypolypeptide comprising an amino acid sequence having at least 25%homology to one of the sequences of SEQ ID NOs.: 7-14, at least 50%homology to one of the sequences of SEQ ID NOs.: 7-14 or at least 80%homology to one of the sequences of SEQ ID NOs.: 7-14. According to adifferent embodiment, the compound is a polypeptide that specificallybinds a γ subunit of the Lipolysis Stimulated Receptor or a gC1q-R or agC1q-R homologue, but the compound is not a subunit of the LipolysisStimulated Receptor. In this instance, the compound includes apolypeptide that can be C1q, AdipoQ, ApM1, Acrp 30, cerebellin ormultimerin, or fragments of any of these polypeptides. In anotherembodiment, the compound that influences the partitioning of dietarylipids between the liver and peripheral tissues can be a polypeptidehaving binding specificity for a γ subunit of the Lipolysis StimulatedReceptor or a gC1q-R or a gC1q-R homologue for the treatment of obesity.In this instance, the polypeptide is not a subunit of the LipolysisStimulated Receptor. According to another embodiment, the polypeptidecan have about 25% homology to an ApM1 protein, about 50% homology to anApM1 protein or about 80% homology to an ApM1 protein. Moreparticularly, the polypeptide can be any of C1q, AdipoQ, ApM1, Acrp 30,cerebellin or multimerin, or fragments of any of these polypeptides.Additionally, the polypeptide can be a human polypeptide, and can be theApM1 polypeptide or a fragment of the ApM1 polypeptide.

Another aspect of the invention relates to a polypeptide thatspecifically binds the gC1q-R protein for use in the treatment ofobesity, wherein the polypeptide is not a subunit of the LipolysisStimulated Receptor. In one embodiment, the polypeptide can be any ofC1q, AdipoQ, ApM1, Acrp 30, cerebellin or multimerin.

A still further aspect of the invention relates to a composition formodulating activity of the Lipolysis Stimulated Receptor. Thiscomposition includes a compound having binding specificity for thegC1q-R protein, but the compound cannot be a subunit of the LipolysisStimulated Receptor. The invented composition also includes apharmaceutically acceptable carrier.

Another aspect of the invention relates to a composition for modulatingactivity of the Lipolysis Stimulated Receptor. This compositionincludes: (1) a polypeptide comprising an amino acid sequence at least25% homologous to a sequence selected from the group consisting of anyone of SEQ ID NOs: 7-14, and a pharmaceutically acceptable carrier.

Still another aspect of the invention relates to a composition formodulating the activity of the Lipolysis Stimulated Receptor andincludes: (1) a polypeptide that includes a consensus sequence that iseither SEQ ID NO:1 or SEQ ID NO:2, and a pharmaceutically acceptablecarrier.

Another aspect of the invention relates to a method of reducing plasmalipoprotein levels in an animal. This method includes the steps of:first identifying an animal having a measurable plasma lipoproteinlevel, then administering to the animal a composition that includes apharmaceutically acceptable carrier and a polypeptide that is at least25% homologous to an ApM1 protein and finally allowing passage of aperiod of time to permit reduction in the measurable plasma lipoproteinlevel. In a particular case the animal is a mammal. In a particularembodiment the composition may be administered by injection, for exampleby injecting intravenously. Alternatively, the composition may beadministered by surgically implanting an infusion device that slowlyreleases the composition.

Another aspect of the invention relates to a method of identifyingcandidate pharmaceutical agents for reducing plasma triglyceride levelsin an animal. This method involves first identifying a compound thatincludes a consensus sequence that may be either SEQ ID NO:1 or SEQ IDNO:2, obtaining a test animal having an initial level of plasmatriglycerides, administering the compound to the test animal, waitingfor a period of time, measuring a post-treatment level of plasmatriglycerides in a blood sample obtained from the test animal andthereafter identifying as candidate pharmaceutical agents any compoundthat results in a post-treatment level of plasma triglycerides that islower than the initial level. In one embodiment the test animal is amammal and the method may involve feeding a high-fat meal to thismammal. The high-fat meal can include about 60% fat, about 20% protein,and about 20% carbohydrate. The fat component may include about 37%saturated fatty acids, about 36% polyunsaturated fatty acids and about36% polyunsaturated fatty acids.

Still another aspect of the invention relates to a method for treatingan animal having a condition in which it is desirable to increase thepartitioning of dietary lipids to the liver. This method includes thestep of administering an LSR agonist to the animal having the condition.

Still yet another aspect of the invention relates to a method fortreating an animal having a condition in which it is desirable todecrease the partitioning of dietary lipids to the liver. This methodincludes the step of administering an LSR antagonist to the animalhaving the condition.

In another aspect, the invention comprises an agent which increases theactivity of a compound which increases the partitioning of dietarylipids to the liver for use as a pharmaceutical. In one embodiment ofthis aspect, the agent is for use in reducing food intake in obeseindividuals, reducing the levels of free fatty acids in obeseindividuals, decreasing the body weight of obese individuals, ortreating an obesity related condition selected from the group consistingof atherosclerosis (whether obesity-related or not), obesity-relatedinsulin resistance, obesity-related hypertension, microangiopathiclesions resulting from obesity-related Type II diabetes, ocular lesionscaused by microangiopathy in obese subjects with Type II diabetes, andrenal lesions caused by microangiopathy in obese subjects with Type IIdiabetes. In another embodiment of this aspect, the agent increases theactivity of adipoQ, ApM1, a compound analogous to adipoQ or ApM1, or theLSR receptor. In a further embodiment of this aspect, the agent isselected from the group consisting of derivatives of adipoQ, ApM1, C1q,derivatives of a compound analogous to any of the preceding compoundswherein the derivatives exhibit greater activity than the correspondingwild type protein and antibodies capable of specifically binding the γsubunit, the C1q receptor (gC1q-R) or a protein related thereto. In yetanother embodiment of this aspect the agent is selected from the groupconsisting of derivatives of compounds comprising at least one of thesequences of SEQ ID NOs.: 1 and 2, derivatives of compounds comprisingan amino acid sequence having at least 25% homology to a sequenceselected from the group consisting of SEQ ID NOs. 7-14, derivatives ofcompounds comprising an amino acid sequence having at least 50% homologyto a sequence selected from the group consisting of SEQ ID NOs. 7-14,and derivatives of compounds comprising an amino acid sequence having atleast 80% homology to a sequence selected from the group consisting ofSEQ ID NOs. 7-14, wherein the derivatives exhibit greater activity thanthe corresponding wild type protein. In still a further embodiment ofthis aspect, the agent comprises a nucleic acid encoding a polypeptideor protein which influences the partitioning of dietary lipids betweenthe liver and peripheral tissues for use as a medicament. In anotherembodiment of this aspect, the nucleic acid encodes a protein orpolypeptide selected from the group consisting of adipoQ, ApM1, C1q,polypeptides analogous to ApM1, polypeptides having at least one of theconsensus sequences of SEQ ID NO:1 and SEQ ID NO:2, analogs of any ofthe preceding polypeptides, homologs of any of the precedingpolypeptides, derivatives of any of the preceding polypeptides, andfragments of any of the preceding polypeptides. In still anotherembodiment of this aspect, the nucleic acid encodes a polypeptideselected from the group consisting of polypeptides comprising an aminoacid sequence having at least 25% homology to one of the sequences ofSEQ ID NOs.: 7-14, polypeptides comprising an amino acid sequence havingat least 50% homology to one of the sequences of SEQ ID NOs.: 7-14, andpolypeptides comprising an amino acid sequence having at least 80%homology to one of the sequences of SEQ ID NOs: 7-14. In a furtherembodiment of this aspect, the agent is selected from the groupconsisting of small molecules and drugs. In yet another embodiment ofthis aspect, the agent is for administration to an individual having abelow normal level of activity of adipoQ, ApM1, or an analoguousprotein.

Another aspect of the present invention is an agent which decreases theactivity of a compound which increases the partitioning of dietarylipids to the liver for use as a pharmaceutical. In one embodiment ofthis aspect, the agent is for use in treating cachexia in subjects withneoplastic or para-neoplastic syndrome or eating disorders. In anotherembodiment of this aspect, the agent decreases the activity of adipoQ,ApM1, a compound analogous to adipoQ or ApM1, or the LSR receptor. In afurther embodiment of this aspect, the agent is an antibody which bindsa compound selected from the group consisting of adipoQ, ApM1, C1q, aprotein analogous to any of the preceding proteins, a derivative ofadipoQ, C1qa, C1qb, C1qc, mul, cer, ApM1, or acrp which inhibits theactivity of wild type adipoQ or wild type ApM1, fragments of any of thepreceding polypeptides, the γ subunit, the C1q receptor (gC1q-R) or aprotein related thereto. In yet another embodiment of this aspect, theagent is an antibody which binds a polypeptide selected from the groupconsisting of polypeptides comprising at least one of the sequences ofSEQ ID NOs.: 1 and 2, polypeptides comprising an amino acid sequencehaving at least 25% homology to a sequence selected from the groupconsisting of SEQ ID NOs.: 7-14, polypeptides comprising an amino acidsequence having at least 50% homology to a sequence selected from thegroup consisting of SEQ ID NOs.: 7-14, and polypeptides comprising anamino acid sequence having at least 80% homology to a sequence selectedfrom the group consisting of SEQ ID NOs. 7-14. In a further embodimentof this aspect, the agent is selected from the group consisting ofantisense nucleic acids to the adipoQ gene, the ApM1 gene or a portionthereof and nucleic acids capable of forming a triple helix with aportion of the adipoQ gene or the ApM1 gene. In yet another embodimentof this aspect, the agent is selected from the group consisting ofantisense nucleic acids to a gene encoding a polypeptide comprising atleast one of the sequences of SEQ ID NOs.: 1 and 2, a gene encoding apolypeptide comprising an amino acid sequence having at least 25%homology to a sequence selected from the group consisting of SEQ ID NOs.7-14, a gene encoding a polypeptide comprising an amino acid sequencehaving at least 50% homology to a sequence selected from the groupconsisting of SEQ ID NOs. 7-14, and a gene encoding a polypeptidecomprising an amino acid sequence having at least 80% homology to asequence selected from the group consisting of SEQ ID NOs. 7-14. In afurther embodiment of this aspect, the agent is selected from the groupconsisting of small molecules and drugs. In a further embodiment of thisaspect, the agent is for administration to an individual having a levelof adipoQ or ApM1 activity which is above normal.

Another aspect of the present invention is a method for determiningwhether an obese individual is at risk of suffering from a conditionselected from the group consisting of a condition associated with alower than desirable level of partitioning of dietary lipids to theliver, obesity-related atherosclerosis, obesity-related insulinresistance, obesity-related hypertension, microangiopathic lesionsresulting from obesity-related Type II diabetes, ocular lesions causedby microangiopathy in obese subjects with Type II diabetes, and renallesions caused by microangiopathy in obese subjects with Type IIdiabetes, comprising the step of determining whether the individual hasa lower than normal level of adipoQ activity, ApM1 activity, or activityof a compound analogous thereto.

Another aspect of the present invention is a method for increasing thepartitioning of dietary lipids to the liver comprising administering anagent which increases the activity of a compound selected from the groupconsisting of adipoQ, ApM1, C1q, compounds analogous to C1q, compoundscomprising at least one sequence selected from the group consisting ofSEQ ID NO:1 and SEQ ID NO:2, compounds comprising an amino acid sequencehaving at least 25% homology to a sequence selected from the groupconsisting of SEQ ID NOs. 7-14, compounds comprising an amino acidsequence having at least 50% homology to a sequence selected from thegroup consisting of SEQ ID NOs. 7-14, and compounds comprising an aminoacid sequence having at least 80% homology to a sequence selected fromthe group consisting of SEQ ID NOs. 7-14 to an individual. In oneembodiment of this aspect, the individual suffers from a conditionselected from the group consisting of obesity, obesity-relatedatherosclerosis, obesity-related insulin resistance, obesity-relatedhypertension, microangiopathic lesions resulting from obesity-relatedType II diabetes, ocular lesions caused by microangiopathy in obesesubjects with Type II diabetes, and renal lesions caused bymicroangiopathy in obese subjects with Type II diabetes. In anotherembodiment of this aspect, the agent is selected from the groupconsisting of a derivative of adipoQ, ApM1 or an analogous compoundwhich exhibits greater activity than the corresponding wild typeprotein, nucleic acids encoding adipoQ, ApM1, or an analogous compound,fragments of any of the preceding compounds, and nucleic acids encodinga derivative of adipoQ, ApM1, or an analogous compound having greateractivity than the corresponding wild type protein, and fragments of anyof the preceding compounds. In a further aspect of this embodiment, theagent is administered if it is determined that the level of ApM1, or ananalogous protein in the individual is below normal.

Another aspect of the present invention is a method for decreasing thepartitioning of dietary lipids to the liver comprising administering anagent which decreases the activity of a compound selected from the groupconsisting of adipoQ, ApM1, C1q, compounds analogous to C1q, compoundscomprising at least one sequence selected from the group consisting ofSEQ ID NO:1 and SEQ ID NO:2, compounds comprising an amino acid sequencehaving at least 25% homology to a sequence selected from the groupconsisting of SEQ ID NOs. 7-14, compounds comprising an amino acidsequence having at least 50% homology to a sequence selected from thegroup consisting of SEQ ID NOs. 7-14, and compounds comprising an aminoacid sequence having at least 80% homology to a sequence selected fromthe group consisting of SEQ ID NOs. 7-14 to an individual. In oneembodiment of this aspect, the individual suffers from a conditionselected from the group consisting of cachexia in subjects withneoplastic or para-neoplastic syndrome or eating disorders. In anotherembodiment of this aspect, the agent is selected from the groupconsisting of an antibody which binds adipoQ, ApM1, C1q or an analogousprotein, a derivative of adipoQ, C1qa, C1qb, C1qc, mul, cer, ApM1, oracrp which inhibits the activity of wild type adipoQ or wild type ApM1,a fragment of the derivative, antisense nucleic acids to the adipoQgene, the ApM1 gene or a portion thereof, nucleic acids capable offorming a triple helix with a portion of the adipoQ gene or the ApM1gene, and antibodies capable of binding the γ subunit, the C1q receptor(gC1q-R) or a protein related thereto. In still another embodiment ofthis aspect, the agent is administered if it is determined that thelevel of adipoq, ApM1, or an analogous protein in the individual isabove normal.

Another aspect of the present invention is a method of identifying acandidate compound for regulating the partitioning of dietary lipidsbetween the liver and the adipose tissue comprising the steps ofcontacting the γ subunit, the C1q receptor (gC1q-R) a protein relatedthereto, or a fragment thereof with one or more molecules to be testedfor binding activity under conditions which permit specific binding ofthe molecule to the γ subunit, C1q receptor (gC1q-R), protein relatedthereto, or fragment thereof and determining whether the one or moremolecules bind to the γ subunit, C1q receptor (gC1q-R), protein relatedthereto, or fragment thereof. In one embodiment of this aspect, thecontacting step is performed using a cell expressing the γ subunit, C1qreceptor (gC1q-R), protein related thereto, or fragment thereof. Inanother embodiment of this aspect, the γ subunit, C1q receptor (gC1q-R),protein related thereto, or fragment thereof is immobilized on asupport. In yet another embodiment of this aspect, the method furthercomprises contacting the γ subunit, C1q receptor (gC1q-R), proteinrelated thereto, or fragment thereof with a known ligand and determiningthe ability of the one or more molecules to be tested for bindingactivity to compete with the known ligand for binding to the γ subunit,C1q receptor (gC1q-R), protein related thereto, or fragment thereof. Ina further embodiment of this aspect, the molecule to be tested forbinding to the γ subunit, C1q receptor (gC1q-R), protein relatedthereto, or fragment thereof is selected from the group consisting ofpolypeptides, peptides, derivatives or analogs thereof, drugs, and smallmolecules.

Another aspect of the invention relates to a method of identifyingcandidate pharmaceutical agents for reducing plasma triglyceride levelsin an animal. This method involves first administering a compound to atest, and measuring a post-treatment level of plasma triglycerides in ablood sample obtained from the test animal. In one embodiment the testanimal is a mammal and the method may involve feeding a high-fat meal tothis mammal. The high-fat meal can include about 60% fat, about 20%protein, and about 20% carbohydrate. The fat component may include about37% saturated fatty acids, about 36% polyunsaturated fatty acids andabout 36% polyunsaturated fatty acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a line graph showing the inhibitory effect of antibodiesdirected against synthetic peptides representing the amino-terminal andcarboxy-terminal regions of the gC1q-R protein. Oleate-induced bindingof ¹²⁵I-LDL to rat hepatocyte plasma membranes was measured in thepresence of increasing concentrations of antibodies directed against anamino-terminal peptide of gC1q-R (▪); antibodies directed against acarboxy-terminal peptide of gC1q-R (◯) or a negative control antibody(□).

FIGS. 2A-2C show line graphs representing different aspects of LSRactivity. The graphs show results for (A) binding, (B) internalization,and (C) degradation of labeled LDL, a model lipoprotein, in the presenceand absence of oleate at increasing concentrations of C1q. Values on thevertical axis are presented in ng of ¹²⁵I-LDL per mg of cellular proteinthat was bound, internalized and degraded per dish in the presence (▪)or in the absence (□) of oleate.

FIG. 3 shows an alignment of several proteins that are analogous to C1q.The globular domains of proteins belonging to the C1q complement familywere aligned using clustalW. The various aligned sequences are:

-   -   C1qa-117: protein sequence of complement C1q A (reference Swiss        Prot: P02745), from the amino acid at position 117 (SEQ ID NO:7)    -   C1qb-122: protein sequence of complement C1q B (reference Swiss        Prot: P02746), from the amino acid at position 122 (SEQ ID NO:8)    -   C1qc-121: protein sequence of complement C1q C (reference Swiss        Prot: P02747), from the amino acid at position 121 (SEQ ID NO:9)    -   mul-1160: protein sequence translated from the nucleic sequence        for multimerin (GenBank, Accession: U27109) from amino acid 1160        (SEQ ID NO:14)    -   cer-64: protein sequence translated from the nucleic sequence        for cerebellin (GenBank, Accession: M58583) from amino acid 64        (SEQ ID NO:10)    -   apm1-115: protein sequence translated from the nucleic sequence        for ApM1 (GenBank, Accession: D45371) from amino acid 115 (SEQ        ID NO:11)    -   adQ-118: protein sequence translated from the nucleic sequence        for AdipoQ (Genbank, Accession: U49915) from amino acid 118 (SEQ        ID NO:12)    -   acrp-118: protein sequence translated from nucleic sequence for        acrp30 (GenBank, Accession: U37222) from amino acid 118 (SEQ ID        NO:13).        Boxed sequences show the two portions of alignment corresponding        to the C1q signature, the first corresponding to the consensus        deposited in the Prosite data base (#PDOC00857):        F-x(5)-[N/D]-x(4)-[F/Y/W/L]-x(6)-F-x (5)-G-x-Y-x-F-x-[F/Y] (SEQ        ID NO:1), the second at the COOH-end of the proteins is:        [S/T]-x-F-[S/T]-G-[F/Y]-L-[L/V]-[F/Y] (SEQ ID NO:2). In these        sequences, the square brackets ([ ]) enclose alternative amino        acids that can occupy a position and numbers indicate the number        of iterations of an unspecified amino acid. The arrows (V) above        the alignments mark the positions of the cystein residues        conserved in the three forms of C1q but not in the other aligned        proteins. The symbols (*) placed under the alignments indicate        the conserved amino acids, the symbols (.) indicate the        conservative substitutions of amino acids.

FIGS. 4A-4C show bar graphs representing different aspects of LSRactivity. The graphs show results for (A) binding; (B) uptake orinternalization; and (C) degradation of ¹²⁵I-LDL by culturedhepatocytes. Open bars represent the difference between values obtainedafter incubation with and without 0.6 mM oleate in the absence ofAdipoQ. Closed bars show the same parameters in samples incubated with25 ng AdipoQ.

FIG. 5 is a line graph showing the postprandial lipemic response in ratsinjected with AdipoQ.

FIGS. 6A-6B are bar graphs representing results obtained followinginfusion of AdipoQ in rats. The graphs show results for (A) weight loss;and (B) plasma triglyceride levels.

FIGS. 7A-7B are bar graphs representing daily food intake for (A) ob/obmice; and (B) db/db mice that were either controls or administered withAdipoQ.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Overview

Herein we disclose compositions and methods that are useful formodulating the activity of the “Lipolysis Stimulated Receptor” (LSR). Asdetailed below, the ability to modulate LSR activity provides a meansfor intervening in pathologies that involve abnormalities in lipidmetabolism. More particularly, we have discovered a family of compoundsthat can be incorporated into medicaments which, when administered invitro or in vivo, advantageously enhance LSR activity. As a consequence,lipoproteins are efficiently bound, internalized and degraded byhepatocytes.

When used in this fashion, compounds that enhance LSR activity,particularly those that enhance receptor activity can promote weightloss. In contrast to LSR-activating compounds, agents that inhibit LSRactivity can be used to promote lipid storage in adipose tissue becauselipoprotein degradation by the liver will be reduced. The inventedcompositions and methods are useful for treating conditions thatinclude: obesity and anorexia, hyperlipidemias, atherosclerosis,diabetes, hypertension, and more generally the various pathologiesassociated with abnormalities in the metabolism of cytokines.

Introduction

The present invention relates generally to methods and compositions thatare useful for regulating the activity of a multi-subunit receptorcalled LSR. The LSR is expressed on the surface of hepatic cells andbinds lipoproteins in the presence of free fatty acids. In the absenceof free fatty acids the LSR can bind a cytokine, preferably leptin.Importantly, the LSR is also capable of binding gC1q-R (Ghebrehiwet etal., J. Exp. Med. 179:1809 (1994)) or a gC1q-R-like receptor. Thosehaving ordinary skill in the art will understand that the gC1q-R is areceptor for C1q, a protein that is a key component of the complementsystem and that is also known to activate phagocytosis by macrophages.

In brief, the LSR includes at least one α and one β subunit, preferablyone α and three β subunits. Both α and β subunits are the translationproducts of two mRNA species that result from alternative splicing of acommon precursor RNA. An α′ (alpha prime) subunit, which is an integralmembrane protein like the α subunit and is encoded by a thirdalternatively spliced mRNA, is believed to be a constituent of LSR inthe alternative to the α subunit. Further inclusion of a γ subunit,which may be gC1q-R or a gC1q-R-like receptor protein, with the LSRresults in the formation of “LSR complex.” We postulate that the gC1q-Ror the gC1q-R like protein serves as a molecular chaperon thatassociates with LSR.

We believe that agents which modify the structure of the LSR complex byperturbing interaction of the γ subunit with the LSR effectivelyactivate the LSR in the absence of free fatty acids. The effect of thisperturbation can be measured as increased hepatocyte binding,internalization and degradation of lipoproteins. When lipids aredegraded within liver cells, fewer lipids are available for uptake andstorage by adipose tissue.

Definitions

As used herein, the terms “LSR” and “LSR receptor” refer to thecombination of α or α′ and β subunits that make up a receptor primarilyexpressed on the surface of hepatocytes and that can bind and facilitatethe internalization and degradation of lipoproteins by hepatocytes.

As used herein, “LSR complex” refers to an LSR receptor which furtherincludes a γ subunit.

As used herein, the term polypeptide is understood to designate aprotein or a peptide.

Equivalent polypeptide will be understood to mean a polypeptide havingat least one of the activities of a subject polypeptide. Thus, forexample, if a subject polypeptide is able to inhibit binding of a γsubunit to the LSR to form an LSR complex, then in this context anequivalent polypeptide will be a polypeptide that similarly is able toinhibit binding of the γ subunit to the LSR.

Homologous polypeptide will be understood to mean polypeptides thatexhibit certain modifications when compared with the naturalpolypeptide. These modifications include a deletion, truncation,extension, chimeric fusion and/or mutation, in particular a pointmutation. Among the homologous polypeptides, those in which the aminoacid sequence exhibits at least 80%, preferably 90%, homology with theamino acid sequences of the polypeptides according to the invention arepreferred.

Derivative polypeptide (or derivative protein) will be understood tomean all the mutated polypeptides which may exist, includingtruncations, deletions and/or additions of amino acid residues(including naturally occurring amino acids, modified and unusual aminoacids such as those listed in Table 4 of WIPO Standard ST.25 (1998), andnon-naturally occurring amino acids), substitutions or mutations, inparticular point mutations, regardless of whether they are naturallyoccurring or whether they have been artificially. Artificially generatedderivates may be created using a variety of techniques, includingmutagenesis of nucleic acids encoding the polypeptides, chemicalsynthesis, or chemical modification.

As used herein the terms “obesity” and “obesity-related” are used torefer to individuals having a body mass which is measurably greater thanideal for their height and frame. Preferably, these terms refer toindividuals with body mass index values of greater than 10, morepreferably with body mass index values of greater than 20, and mostpreferably with body mass index values of greater than 35.

Polypeptide fragment is understood to mean a polypeptide or a peptidecomprising at least 5, at least 7, at least 10, at least 15, at least30, or more than 30 consecutive amino acids of the polypeptide fromwhich they are derived. It will be understood that a polypeptidefragment may be obtained from a derivative polypeptide.

Biologically active fragments of a polypeptide will be understood tomean a portion of a larger polypeptide wherein said portion retains anactivity characteristic of the larger polypeptide, and wherein theactivity is measurable in any biological system. For example, apolypeptide fragment is deemed to be “biologically active” if itdemonstrates a statistically significant change in activity in any ofthe assays described in Examples 1, 2, 6, 7, 8, 9 or 10. Thus, forexample, if a protein characteristically modifies the interactionbetween the γ subunit and the LSR receptor, then a biologically activefragment of that protein would be a portion of the protein that retainsthe ability to modify said interaction.

A polynucleotide, nucleic sequence or nucleic acid is understood to meanan isolated natural or synthetic DNA and/or RNA molecule which mayinclude non-natural nucleotides.

Equivalent polynucleotide sequences are understood to mean nucleic acidsequences encoding the polypeptides according to the invention, takinginto account the degeneracy of the genetic code, the complementary DNAsequences and the corresponding RNA sequences, as well as the nucleicacid sequences encoding the equivalent polypeptides.

Homologous nucleic sequences are understood to mean the nucleicsequences encoding the homologous polypeptides and/or the nucleicsequences exhibiting a level of homology of at least 80%, preferably90%. According to the invention, the homology is only of the statisticaltype, it means that the sequences have a minimum of 80%, preferably 90%,of nucleotides in common.

Allele or allelic variant will be understood to mean the natural mutatedsequences corresponding to poly-morphisms present in human beings and,in particular, to polymorphisms which can lead to the onset and/or tothe development of obesity or of anorexia. These polymorphisms can alsolead to the onset and/or to the development of risks or complicationsassociated with obesity, in particular at the cardiovascular level,and/or of pathologies associated with abnormalities in the metabolism ofcytokines.

Mutated nucleic sequences are understood to mean the nucleic sequencescomprising at least one point mutation compared with the normalsequence.

While the sequences according to the invention are in general wild typesequences, they are also mutated sequences since they comprise at leastone point mutation and preferably at most 10% of mutations compared withthe wild type sequence.

As referred to herein, methods and medicaments of the invention can beused for treating animals, including birds, fish and mammals. It is tobe understood that the category of mammals includes mammals such asmice, rats, rabbits, domesticated mammals and human beings. Although themethods of treatment can be applied to non-human mammals, we clearlyenvision that humans can also be treated using the methods andmedicaments disclosed herein.

LSR Activity-Modulating Compounds

Using the methods disclosed herein, compounds that selectively modulatethe activity of the LSR in vitro and in vivo have been identified. Thecompounds identified by the process of the invention include, forexample, antibodies having binding specificity for the gC1q-R protein,C1q and AdipoQ. Since ApM1 is reasonably expected to represent the humanhomologue of murine AdipoQ, as described below, it follows that ApM1will be useful for modulating LSR activity and lipoprotein metabolism inhumans. More generally, it is expected that homologues of C1q will beuseful for modulating LSR activity and lipoprotein metabolism. Thecompounds of the present invention, however, are not limited to anyparticular chemical structure, as they are solely defined by the assaycascade of the invention, which allows, for the first time, forsystematic and rational identification of highly potent and selectivemodulators of the hepatocyte-specific LSR on a molecular level.

Indications

While not wishing to be bound by any particular theory of operation, thecompounds identified by the methods of the present invention arebelieved to bind to the γ subunit of the LSR complex whereat thecompound either will enhance or inhibit LSR activity. Thus,pharmaceutical compositions comprising a therapeutically effectiveamount of a compound identified by the process of the invention will beuseful for the treatment of diseases characterized by high levels ofcirculating triglycerides or an undesirably strong tendency for lipiddeposition at adipose tissue. Alternatively, compounds that inhibit LSRactivity will be useful for favoring lipid deposition to the adiposetissue and/or diminishing liver degradation of dietary lipids.

Thus, in general, the disorders which may be treated with the compounds,compositions, medicaments and pharmaceutical formulations identified bythe process of the invention generally refer to disorders involvinglipid metabolism.

Pharmaceutical Formulations and Routes of Administration

The identified compounds can be administered to a mammal, including ahuman patient, alone or in pharmaceutical compositions where they aremixed with suitable carriers or excipient(s) at therapeuticallyeffective doses to treat or ameliorate a variety of disorders associatedwith lipid metabolism. A therapeutically effective dose further refersto that amount of the compound sufficient to result in amelioration ofsymptoms as determined by the methods described herein. Thus, atherapeutically effective dosage of AdipoQ or ApM1 will be that dosageof the compound that is adequate to promote reduced triglyceride levelsfollowing a high-fat meal and that will promote weight loss withcontinued periodic use or administration. Techniques for formulation andadministration of the compounds of the instant application may be foundin “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton,Pa., latest edition.

Routes of Administration.

Suitable routes of administration include oral, rectal, transmucosal, orintestinal administration, parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal orintraocular injections. A particularly useful method of administeringcompounds for promoting weight loss involves surgical implantation, forexample into the abdominal cavity of the recipient, of a device fordelivering the compound over an extended period of time. Sustainedrelease formulations of the invented medicaments particularly arecontemplated.

Composition/Formulation

Pharmaceutical compositions and medicaments for use in accordance withthe present invention may be formulated in a conventional manner usingone or more physiologically acceptable carriers comprising excipientsand auxiliaries. Proper formulation is dependent upon the route ofadministration chosen.

Certain of the medicaments described herein will include apharmaceutically acceptable carrier and at least one polypeptide that ishomologous to the C1q protein or a fragment thereof. In addition tomedicaments that include protein components homologous to the C1qprotein homologues, we also contemplate that non-protein compounds thatinteract with the γ subunit of the LSR complex also will find utility asmodulators of LSR activity, both in vitro and in vivo. Included amongexamples of C1q protein homologues that will find utility in modulatingLSR activity and/or stimulating a reduction of plasma lipoproteinsand/or promoting weight loss are: the C1q proteins (C1q A, C1q B and C1qC), AdipoQ, ApM1, acrp 30, cerebellin and multimerin.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer suchas a phosphate or bicarbonate buffer. For transmucosal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillerssuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable gaseous propellant, e.g., carbon dioxide. In the case of apressurized aerosol the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin, for use in an inhaler or insufflator, may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form. Aqueoussuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder or lyophilizedform for constitution with a suitable vehicle, such as sterilepyrogen-free water, before use.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various sustained release materialshave been established and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days.

Depending on the chemical nature and the biological stability of thetherapeutic reagent, additional strategies for protein stabilization maybe employed.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Effective Dosage.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve their intended purpose. More specifically, atherapeutically effective amount means an amount effective to preventdevelopment of or to alleviate the existing symptoms of the subjectbeing treated. Determination of the effective amounts is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes orencompasses a concentration point or range shown to effect enhanced orinhibited LSR activity in an in vitro system. Such information can beused to more accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms in a patient. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50, (the dose lethal to 50% of the testpopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio between LD50and ED50. Compounds which exhibit high therapeutic indices arepreferred.

The data obtained from these cell culture assays and animal studies canbe used in formulating a range of dosage for use in human. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50, with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See, e.g.,Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active compound which are sufficient to maintainthe LSR modulating effects. Dosages necessary to achieve the LSRmodulating effect will depend on individual characteristics and route ofadministration.

Dosage intervals can also be determined using the value for the minimumeffective concentration. Compounds should be administered using aregimen which maintains plasma levels above the minimum effectiveconcentration for 10-90% of the time, preferably between 30-90%; andmost preferably between 50-90%. In cases of local administration orselective uptake, the effective local concentration of the drug may notbe related to plasma concentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

A preferred dosage range for the amount of polypeptide homolog of C1q,such as AdipoQ or ApM1, that can be administered on a daily or regularbasis to achieve desired results, including a reduction in levels ofcirculating plasma triglycerides and/or lipoproteins, range from 0.1-50mg/kg body mass. A more preferred dosage range is from 0.2-25 mg/kg. Astill more preferred dosage range is from 1.0-20 mg/kg, while the mostpreferred range is from 2.0-10 mg/kg. Of course, these daily dosages canbe delivered or administered in small amounts periodically during thecourse of a day.

Protein Homologies

It is to be understood that a polypeptide having a given level ofhomology to a subject protein or polypeptide can be identified usingreadily available sequence alignment and comparison programs, such asblastp, fasta and/or ClustalW, and methods that will be familiar tothose having ordinary skill in the art. One approach for identifying aprotein that is homologous to a subject protein involves running astandard “blastp” (Altschul et al., J. Mol. Biol. 215:403-410 (1990)sequence comparison algorithm. In this approach, a relatively low scorein the blastp algorithm can be used to isolate large numbers ofpotentially homologous sequences of different lengths. For example, alow score may be on the order of from between about 80 to about 100 andmay result in targets having homology levels of about 20%.

If higher levels of homology are desired, additional steps can be taken.For example, once a first series of candidate homologous sequences hasbeen identified, sequences that are more homologous to the subjectprotein can then be selected. At that stage, two parameters can bevaried. First, it is possible to “cut” the subject protein intosub-sequences of interest and then run homology searches that refine theresults obtained in the initial step. For example, the fragments thatare listed as SEQ ID 7-14 or the consensus sequences within the twoboxed regions shown in FIG. 3 could be selected as sub-sequences ofinterest that could be used to run homology searches. Second, the scoreused in running the blastp algorithm can be increased. For example, byincreasing the score up to a level of about 300, homology levels ofabout 80% frequently can be obtained. These procedures can be carriedout using easily accessible computer programs such as, for example,“blastp” or “fasta”. (Altschul et al., J. Mol. Biol. 215:403-410(1990);Pearson, W. R. Genomics 11:635-650 (1991)).

Those having ordinary skill in the art will appreciate that theabove-referenced score that can be input into the sequence comparisonprogram can be calculated based on the degree of homology that is beingsought. Formulas within the cited packages of computer algorithms allowfor this possibility. In general an increasing score allows for theidentification of increasingly specific protein alignments characterizedby high levels of homology. Candidate alignments can be further refinedusing pairwise (fasta) or multiple alignments (ClustalW) (Higgins etal., Computer Applications in the Biosciences (CABIOS), 8:189-191 (1992)and Thompson et al., Nucleic Acids Research 22:4873-4680 (1994)).

An illustration of the extent of protein homology appears in Table 1.The results appearing in the table show various homology levels betweenthe ApM1 and various whole proteins or fragments of those proteins.Thus, for example, the entire Acrp 30 protein sequence exhibited 81.8%homology with the ApM1 protein, while the Acrp 30 segment identified bySEQ ID NO:13 showed 91.5% homology with the ApM1 protein.

TABLE 1 Homologies Between ApM1 and Various Proteins Protein/ SEQ ID NO:Whole protein Seq ID Nos: 7-14 Acrp 30/ 81.8% 91.5% SEQ ID N0: 13AdipoQ/ 80.6%   90% SEQ ID NO: 12 C1qa/ 32.9% 27.1% SEQ ID NO: 7 C1qb/31.8% 36.7% SEQ ID NO: 8 C1qc/ 38.8%   38% SEQ ID NO: 9 Multimerin/27.7% 28.8% SEQ ID NO: 14 Cerebellin/ 24.6% 28.3% SEQ ID NO: 10

In order to identify a protein having a given level of homology, such asat least 25% homology, at least 50% homology, or at least 80% homologyto another protein, such as AdipoQ, ApM1 or C1q, one can use a standardblastp analysis in which the program is instructed to recover proteinshaving a score corresponding to the desired level of homology. Forexample, to identify proteins having a homology level of 20-30% theprogram may be instructed to recover proteins having a score frombetween about 80 to about 100. To recover a protein having an 80%homology level, one can instruct the program to include proteins havinga score of about 300. It will be appreciated that these scores may becomputed over the full length of the subject protein or over a portionof the protein such as SEQ ID NOs 7-14. It will further be appreciatedthat homology levels other than those explicitly enumerated herein canbe obtained using the instructions provided as part of the program.Thus, the foregoing description is adequate to allow one of ordinaryskill in the art to identify polypeptides that are homologous to asubject protein, such as the human ApM1 protein, at various levels ofhomology. In some cases, the default parameters may be used.

The Multi-subunit LSR

The hepatocyte-specific LSR is a multi-subunit receptor having a dualactivity. When activated by free fatty acids, the LSR allows endocytosisof lipoproteins and so is a component in a metabolic pathway for theclearance of lipoproteins. This pathway serves mainly, but notexclusively, to promote clearance of particles high in triglycerides ofintestinal origin (Mann et al., Biochemistry 34:10421 (1995)). Thisactivity, expressed most particularly at the hepatic level, is dependenton the presence of free fatty acids which bind to the receptor, induce areversible change in the conformation of this complex and allow it tobind, with a high affinity, various classes of lipoproteins such asthose containing apoprotein B or apoprotein E. In its other role, and inthe absence of free fatty acids, the LSR does not bind lipoproteins, butis capable of binding a cytokine, in particular leptin. Thereceptor-bound leptin is then internalized by the hepatocyte where it isdegraded.

As described above, the LSR subunits can bind gC1q-R, the receptor forC1q, one of the components of the complex C1 of the conventional pathwayfor complement activation. Proteins analogous to gC1q-R also can bind atthe LSR site for binding of gC1q-R. Proteins analogous to gC1q-R areunderstood to mean in particular the homologous proteins preferablyexhibiting a level of amino acid sequence homology of at least 80%, theproteins exhibiting at least one of the motifs of a site for binding ofthe protein gC1q-R on the LSR receptor and/or the proteins capable ofinteracting with the LSR receptor.

Another feature of the LSR relates to the fate of bound lipoproteins orcytokines. More particularly, it is a characteristic of the LSR thatbound lipoproteins or bound cytokines are incorporated into the cell andthen degraded. The bound lipoproteins may particularly containapoprotein B or E.

As described in supplemental detail below, activity of the LSR complexcan be modulated by a family of compounds that includes C1q or one ofits analogous compounds, such as AdipoQ (Hu et al., J. Biol. Chem.271:10697 (1996)), ApM1 (Maeda et al., Biochem. Biophys. Res. Commun.221:286 (1996)) and cerebellin. In particular, the compound C1q or oneof its analogous compounds makes it possible, in the absence of freefatty acid, to enhance LSR activity and so to increase the quantity oflipoproteins bound, internalized and degraded by the cells thatexpresses the LSR receptor.

The invention relates in part to polypeptides or other compounds thatare able to modulate, either by mimicking, promoting, inhibiting orotherwise altering the interaction of the γ subunit of the LSR complexwith the α or α′ and β subunits of the LSR receptor.

The polypeptides of the invention are obtained by purification orisolation from natural sources or alternatively obtained by geneticrecombination, or chemical synthesis. In the case where the polypeptidesare synthetic polypeptides, they can contain non-natural amino acids.

A more complete definition of the invention can be made by firstdescribing the structure of the LSR and the methods that were used toelucidate its pharmacological properties.

Defining the Structure and Function of the LSR

The LSR subunits were identified by procedures that employedreceptor-specific polyclonal antibodies. These antibodies were obtainedby immunizing rabbits with a gel-purified 240 kDa species that bound alabeled LDL probe in a ligand blotting assay (Mann at al., Biochemistry34:10421 (1995)) conducted in the presence of 0.8 mM oleate. In theprocedures described herein, oleate is employed as a model free fattyacid used for activating the lipoprotein binding activity of the LSR.Results of this ligand blotting assay further indicated that membraneproteins having estimated molecular weights of 115 kDa and 90 kDa alsobound LDL. Notably, the rabbit antibodies recognized all three of theLDL-binding species in a Western blotting procedure, and inhibited thebinding of LDLs to LSRs disposed on rat hepatocytes in a ligand bindingassay. This indicated that the polyclonal antibodies raised against the240 kDa LDL-binding species were useful as LSR-specific reagents.

Electrophoretic analysis of proteins that were immunoprecipitated usingthe anti-LSR antibodies and then separated under reducing conditionsindicated that only a very small number of individual proteins wererecognized by the antibody reagent. More particularly, it was found thatproteins of 68 kDa, 56 kDa and 35 kDa were present in theimmunoprecipitate. This demonstrated that the LSR complex was a multimercomposed of subunits having molecular weights of 68 kDa (α subunit), 56kDa (β subunit) and 35 kDa (γ subunit). These molecular weights areestimates and were obtained from the rat. As detailed below, the γsubunit is believed to correspond to a previously known protein. Thestructures of the α and β subunits were established by an expressioncloning procedure using the polyclonal anti-LSR antibodies as a probe.

Indeed, a λgt11 rat liver cDNA library was screened with theLSR-specific antibodies to identify clones expressing proteinscorresponding to the LSR subunits. Detailed analysis of one of theisolated phage clones using a PCR protocol based on the identifiedcloned sequence led to the identification of three different mRNAspecies. Careful examination of the sequences of these mRNAs, togetherwith subsequent PCR and cloning procedures, confirmed the three speciesrepresented alternative splicing variants of a single precursortranscript. The three complete cDNAs had lengths of 2097 bp, 2040 bp and1893 bp. The molecular weights of the predicted proteins encoded by theopen reading frames in the three cDNA sequences were 66 kDa, 64 kDa and58 kDa, respectively.

Northern blotting procedures using RNA isolated from different rattissues indicated that the cloned polynucleotide hybridized totranscripts expressed in rat liver as 1.9 kb and 2.1 kb mRNAs, andfurther indicated that this expression was substantially restricted toliver.

Five different techniques were used to demonstrate that the 2097 bp andthe 1893 bp were essential components of the LSR receptor. First,polyclonal antibodies were raised against two synthetic peptides havingsequences corresponding to residues 169-186 and to residues 556-570respectively, encoded by the 2097 bp cDNA. These peptide sequences werecommon to all of the predicted proteins encoded by the three mRNA splicevariants described above. It was shown that these antipeptideantibodies, but not irrelevant control antibodies, inhibited the bindingof LDLs to the LSR present on rat plasma membranes. Second, Westernblotting and ligand blotting procedures showed that partially purified αand β subunits: (1) bound the rabbit polyclonal anti-LSR antibodies; (2)bound the anti-peptide antibodies, and (3) bound LDLs after incubationwith oleates. Third, in vitro translation and labeling to producesynthetic proteins corresponding to the polypeptides encoded by the 2097bp and the 1893 bp cDNAs led to products that bound LDL in anultracentrifugation assay in which LDL binding produced a complex havinga density that was lower than the density of the protein alone. Thisbinding was enhanced in the presence of oleate by two fold for the αsubunit, or by five fold for the β subunit. Fourth, Chinese hamsterovary cells (CHO) transiently transfected with a plasmid containing theLSR a subunit were found to display an increased binding of LDL afterincubations in the presence of oleate while LDL binding measured in theabsence of oleate remained unchanged. Cotransfection of plasmidscontaining the β LSR subunit together with the α LSR subunit furtherincreased the binding of LDL observed after incubations with oleate.Most importantly, an increase in LDL degradation was observed afterincubation with oleate only in dishes containing cells cotransfectedwith the LSR α and β subunits. Therefore, cotransfection of both LSR αand β subunits is a condition sufficient to increase LSR activity in CHOcells. The affinity of the various classes of lipoproteins for the LSRin cells cotransfected with the α and β subunit was very similar to thatoriginally described for the LSR expressed in rat hepatocytes or inhuman fibroblasts isolated from subjects with familialhypercholesterolemia (Bihain et al., Biochemistry 31:4628 (1992); Yen etal., Biochemistry 33:1172 (1994)).

A fifth line of evidence demonstrating that the identified gene wasresponsible for LSR function and that this receptor participates in theclearance of dietary triglycerides was obtained using genetically obesemice. Both ob/ob mice (having a deficient leptin gene) and db/db mice(having a defect in the gene encoding the leptin receptor) were found toexhibit an increased postprandial lipemic response after forced feedingof the standard test meal described above. The apparent number of LSRavailable on the plasma membrane of hepatocytes of these obese mice waslower than that of the lean controls. Further, analysis by Northernblotting revealed that the level of LSR mRNA was reduced significantlyin obese mice. Treatment of the obesity of ob/ob mice by dailyadministration of recombinant leptin over a thirty day period led tomore than a 30% reduction of the animal body weight; to a massivedecrease in the postprandial lipemic response; to a significant increasein the apparent number of LSR expressed at the surface of liver cellsand to an increased number of LSR mRNA.

On the basis of these data it was therefore established that theidentified LSR gene was responsible for the function of this receptor;that the LSR represents a rate limiting step for the clearance ofdietary triglycerides and that the expression of this gene isdisregulated in obese mice. Moreover, the LSR 56 and 68 kDa subunitssubstantially bound LDLs only after incubation with oleate.Stoichiometric analysis of immunoprecipitation products indicated thatthe 240 kDa LDL-binding complex that was observed in the ligand blottingassay most likely represented a multimeric complex formed by a single αand three β subunits. It is believed that the above-described 2040 bpcDNA which represents the third alternative RNA splicing product encodesa subunit that can substitute for α in the multimeric complex. Thislatter protein is referred to as α′ (alpha prime).

While the α and β subunits of LSR were encoded by alternatively splicedmRNAs generated from a single precursor molecule, the expression cloningprocedure described above did not provide insight into the identity ofthe 35 kDa subunit that was detected along with the 68 kDa and 56 kDaproteins in the anti-LSR immunoprecipitate Indeed, the γ subunit of theLSR was identified by direct sequencing of the purified protein.

More particularly, N-terminal sequencing of immunoaffinity purifiedmaterial was used to establish the likely identity of the finalcomponent of the LSR complex. In this procedure column-immobilizedpolyclonal rabbit anti-LSR antibodies were used to capture membraneproteins from rat liver. After verifying the presence of the 35 kDaspecies in the column eluate, a sample containing the 35 kDa protein wassequenced using a standard Edman degradation protocol. Results from thisprocedure gave a 19 amino acid long polypeptide sequence that was usedto search a protein data base. This search revealed that the γ subunitof the LSR receptor included a polypeptide sequence that identicallyappeared in gC1q-R (Ghebrehiwet et al., J. Exp. Med. 179:1809 (1994)), aknown cell surface receptor that binds the globular heads of C1q. Sincethe entire sequence of the immunoaffinity purified 35 kDa protein wasnot established, we allow the possibility that the γ subunit of the LSRcomplex is related, but not identical to the gC1q-R protein.

Analysis of the protein sequences of the α and β subunits of the LSRrevealed several interesting structural features. For example, thepresence of several phosphorylation sites at the N-terminal end of the αsubunit protein suggested that the amino terminus of this protein wasoriented toward the inside of the cell, and further suggested a possiblerole in signal transduction. The N-terminal portion of the α subunitprotein also possessed a hydrophobic amino acid sequence that wasseparated by two contiguous proline residues, an arrangement likely toinduce a hairpin structure. This arrangement of two hydrophobic armslikely constitutes a putative fatty acid binding domain of the LSR. Theα subunit also possessed a hydrophobic amino acid sequence consistentwith a potential transmembrane domain (Brendel et al., Proc. Natl. Acad.Sci. USA 89:2002 (1992)). The β subunit protein does not possess atransmembrane domain and is probably positioned outside of the cellwhere it is bound through disulfide bridges to other components of theLSR complex.

Compositions and Methods for Modulating LSR Activity

An additional structural feature of the α and β subunit proteins relatedto the presence of repeated segments that were rich in serine andarginine residues. This was significant because the lamin receptor and“splicing factor 2” also have in common a repeated sequence of serineand arginine residues (RSRS), and these proteins also are known tocombine with the gC1qR protein (Honoré et al., Gene 134:283 (1993)). Inview of this coincidence of related structural motifs and interactionswith gC1q-R, we speculated that the serine and arginine rich segments ofthe LSR α and β subunits were somehow important for contact with gC1q-R,or the gC1q-R-like protein that was the γ subunit of the LSR complex.

As described in the following Example, polyclonal antibodies directedagainst synthetic peptides derived from the gC1q-R primary amino acidsequence were used to demonstrate that this protein, or a proteinclosely related to gC1q-R, was a component of the LSR complex. In theprocedure described below, the anti-peptide antibodies inhibited thebinding of labeled LDL to the LSR expressed on the surface of rathepatocytes. Use of the LDL model substrate in these procedures provideda convenient and highly sensitive means for monitoring aspects oflipoprotein metabolism in liver cells.

Example 1 describes the procedures used to demonstrate that gC1q-R, or aclosely related homologue of this protein, was a constituent of the LSRcomplex.

EXAMPLE 1 The gC1q-R or a gC1q-R-like Protein is a Component of theMulti-subunit LSR

Rabbit polyclonal antibodies directed against two synthetic peptideshaving sequences located within the carboxy- and amino-terminal ends ofthe gC1q-R protein were prepared according to standard laboratoryprocedures. The synthetic peptide representing the N-terminal region ofthe protein had the sequence, LRCVPRVLGSSVAGY* (SEQ ID NO:3) andcorresponded to residues 5-19 of the gC1q-R polypeptide sequence. TheC-terminal synthetic peptide had the sequence, C*YITFLEDLKSFVKSQ (SEQ IDNO:4) and corresponded to residues 268-282 of gC1q-R. Amino acidpositions marked with “*” indicate residues that differed from the wildtype protein sequence in order to enhance peptide antigenicity. Peptideswere coupled to a keyhole limpet hemocyacin (KLH) carrier prior toinjection into rabbits. These procedures resulted in two serum samples,each with a binding specificity for a different region of the gC1q-Rprotein. Immunoglobulin G (IgG) from these sera were further purifiedusing a Protein A column (Pharmacia) according to the manufacturer'sinstructions.

Increasing amounts of the these anti-peptide antibodies or an irrelevantIgG antibody were combined with ¹²⁵I-LDL in a standard assay formeasuring oleate-induced binding of LDL to plasma membranes of rathepatocytes (Bihain et al., Biochemistry 31:4628 (1992); Mann et al., J.Biol. Chem. 272:31348 (1997)). The binding induced by oleate wasdetermined as the difference between incubations with and without 0.5 mMoleate. Numerical measurements in this experiment were analyzed as thepercent of total ¹²⁵I-LDL bound to membranes in the absence of addedantibodies.

The results presented in FIG. 1 indicated that antibodies directed toeither of two regions of the gC1q-R protein inhibited LSR activity asmeasured by LDL binding. The negative control IgG did not inhibit LSRactivity in this assay. This proved that inhibitory effects observed inour procedures were the results of specific antibody-receptorinteractions. These results confirmed that gC1q-R, or a protein closelyrelated to gC1q-R, was a component of the LSR complex.

The foregoing results seemingly suggested that agents which bound thegC1q-R, or the γ subunit of the LSR complex, had a negative orinhibitory effect on LSR activity. Thus, we had identified agents thatwere able to modulate LSR activity in a negative way.

In view of these findings, it was of interest to further evaluate theeffects of compounds that bound to gC1q-R, or that might alterinteractions between the γ subunit and the LSR. Since it was known thatthe C1q complement protein was a binding substrate for gC1q-R, weinvestigated whether C1q would modulate LSR activity in the same waythat anti-peptide antibodies inhibited LSR activity in the precedingExample. Notably, the experiment described in the following Example wasconducted both in the presence and absence of oleate, a free fatty acidthat unmasks the lipoprotein binding site on the LSR.

Example 2 describes the procedures used to demonstrate that LSR activitycould be modulated in a positive manner. Unexpectedly, enhancement ofLSR activity took place both in the presence and absence of free fattyacids.

EXAMPLE 2 Regulation of LSR Activity by C1q and its Homologues

Primary cultures of rat hepatocytes were incubated with 20 ng ofleptin/well using 6-well plates for 30 minutes at 37° C. in order tostimulate mobilization of LSR proteins to the cell surface and toincrease the number of LSR receptors expressed. Increasingconcentrations of C1q (Sigma) and 20 μg/ml of ¹²⁵I-LDL were then addedto parallel cell cultures in the presence or absence of 0.5 mM oleate.The mixtures were then incubated 4 hours at 37° C. and the binding,internalization and degradation of the labeled LDL analyzed usingstandard techniques (Bihain et al., Biochemistry 31:4628 (1992); Mann etal., J. Biol. Chem. 272:31348(1997)).

The results presented in FIGS. 2A-C unexpectedly indicated that C1qenhanced LSR activity both in the presence and absence of free fattyacids. Indeed, it was surprising that lipoprotein binding,internalization and degradation occurred in the absence of added oleatebecause these aspects of LSR activity were previously thought to requirethe presence of free fatty acids. Small but meaningful increases in allthree of the measured parameters also were observed in the presence ofoleate. The significance of these latter increases was less substantialbecause the background values measured in the absence of added C1q werehigher in the presence of oleate compared to the values measured in theabsence of this free fatty acid.

The results described in the proceeding Example showed that incubationof rat hepatocytes with C1q, a protein capable of binding gC1q-R andhence potentially capable of displacing it from the LSR complex, led tospontaneous activation of the LSR in the absence of free fatty acids.While not wishing to be bound by any particular theory which underliesthe mechanism of this receptor modulation, we offer the following as apossible explanation for the phenomenon. It is possible that the gC1q-Rprotein, or more generally the γ subunit, functions as a chaperonprotein for the LSR. It is further possible that the γ subunit somehowexerts an inhibitory effect on the LSR. Conceivably then, agents whichperturb or alter the binding of the γ subunit to the LSR can be used tomodulate LSR activity which can be measured in vitro as the binding,internalization and degradation of LDLs.

The exemplary case presented above suggested that C1q served as theagent that perturbed binding of the γ subunit in the LSR complex.However, we contemplate that any agent homologous or analogous to C1qthat is able to bind gC1q-R or a gC1q-R-like protein also will have theeffect of modulating LSR receptor activity.

The above-described effect of C1q on the activity of LSR led us toinvestigate whether similar effects on LSR would be promoted by proteinssharing structural homology with C1q. Alignments for some of thesehomologues are presented in FIG. 3, with the boxed regions representingconserved regions of structural homology. The murine proteins AdipoQ (Huet al., J. Biol. Chem. 271:10697 (1996)) and Acrp30 (Scherer et al., J.Biol. Chem. 270:26746 (1995)), and the human ApM1 protein (Maeda et al.,Biochem. Biophys. Res. Commun. 221:286 (1996)) clearly exhibit markedhomologies. These three proteins, like the components of complement C1q(C1q A, B and C), are secreted proteins having N-terminal ends whichresemble collagen (repetition of Gly-X-Y motifs), and C-terminal endscorresponding to the globular domain of complement C1q. Significantly,these three proteins are preferentially expressed in adipose tissue.Other protein homologues exhibit globular domains resembling the C1qdomain. More specifically, cerebellin and multimerin (isolated in man),are two proteins that do not have a domain which resembles collagen.

Interestingly, conserved cysteine residues at positions 172, 179, 178and 190, 196, 192 respectively in C1q A, C1q B and C1q C are notconserved in the other C1q homologues shown in the alignment. Thesecysteine residues are replaced in ApM1, AdipoQ and Acrp 30, by a lysineresidue and an aspartate residue. Those having an ordinary level ofskill in the art will appreciate that lysine and aspartate amino acidscan, under appropriate conditions, form intrachain salt bridges whichmay contribute to protein structure. The amino acids at correspondingpositions in cerebellin and multimerin would not allow for the formationof salt bridges. It is therefore possible to characterize the C1q domainof the proteins produced by the adipocytes by the absence of cysteinesin the region corresponding to amino acids 170-200 of the molecules ofC1q and by the consensus in the C1q domain.

When considering the structural relationship of the homologues presentedin FIG. 3 it is worth noting that the protein ApM1, which is encoded byan mRNA characterized as being strongly expressed in adipocytes,exhibits 79.7% nucleic acid identity and 80.6% amino acid identity withAdipoQ. Given this level of sequence relatedness, the ApM1 protein isalmost certainly the human homologue of murine AdipoQ. Thus, it is areasonable expectation that the activities of murine AdipoQ which aredisclosed below also will characterize ApM1 in a human system.

Given that C1q has a broad spectrum of biological effects, includinginitiation of the complement cascade, it seemed unlikely that the highlyspecialized activation of the LSR represented a physiologicallysignificant function of this protein. Accordingly, we investigatedwhether C1q homologues could modulate LSR activity. As indicated in theExamples which follow, we have now demonstrated that AdipoQ, an abundantplasma protein having a heretofore unknown function, also enhances LSRactivity.

AdipoQ is a C1q homologue that is known to be secreted by adipocyteswith kinetics closely resembling to the kinetics of Adipsin secretion.Adipsin is a hormone of the complement system and has been shown tocorrespond to the purified fragment of the third component ofcomplement, C3a-desArg (Baldo et al., J. Clin. Invest. 92:1543 (1993)).Adipsin stimulates adipocyte triglyceride synthesis and regulatespost-prandial lipemia (Sniderman et al., Proc. Nutr. Soc. 56:703(1997)). Moreover, secretion of both AdipoQ and Adipsin is stimulated inresponse to insulin.

As supported by the experimental results presented below, we have provedthat AdipoQ can stimulate LSR activity in vitro, and can decrease animalbody weight. Since C1q and AdipoQ share structural homology without alsosharing extensive functional similarities, our demonstration that AdipoQactivates LSR activity establishes the general utility of C1q homologuesas compounds useful for modifying the activity of the LSR.

Example 3 describes the methods used to prepare an expression vectorencoding murine AdipoQ.

EXAMPLE 3 Construction of an Expression Vector Encoding Murine AdipoQ

Standard laboratory procedures were used to isolate RNA from adiposetissue that had been obtained from C57BL/6J mice. Poly(A)* mRNA wascaptured using oligo-dT coated magnetic beads according to themanufacturer's instructions (Dynal, France). The mRNA was reversetranscribed into cDNA using SUPERSCRIPT reverse transcriptase andreagents that were purchased as a kit (Life Technologies, France). cDNAencoding AdipoQ was amplified in a standard PCR protocol usingoligonucleotide primers having the sequences:CTACATGGATCCAGTCATGCCGAAGAT (SEQ ID NO:5), andCGACAACTCGAGTCAGTTGGTATCATGG (SEQ ID NO:6). This procedure selectivelyamplified polynucleotide sequences downstream of the putative signalsequence located at the 5′ end of the AdipoQ coding region. Theamplified cDNA was digested with BamHI and Xhol restrictionendonucleases and the digestion products ligated into the correspondingsites of the pTRC His B expression vector (Invitrogen, France). Thisvector has been engineered to permit expression of heterologoussequences downstream of a polypeptide domain which includes ahexahistidine peptide motif, an enterokinase cleavage site and anepitope that is recognized by an Anti-Xpress™ antibody. Followingtransformation of competent DH5-α E. coli, bacterial clones harboringthe polynucleotide encoding AdipoQ were selected by growth in thepresence of ampicillin. Plasmid DNA was isolated from one of thebacterial clones and the sequence of the heterologous DNA insertdetermined. The sequence of the insert was found to correspond to bases57-762 of AdipoQ (GenBank accession No. U49915). The clonedpolynucleotide sequence also corresponded to bases 86-791 of thesequence encoding Acrp30 (GenBank accession No. U37222), except fornucleotide position 382. The polynucleotide sequence encoding Acrp30 hasan adenosine residue at this position while the AdipoQ polynucleotidehas a guanine residue at the corresponding position. This nucleotidesubstitution leads to an amino acid change from a methionine in Acrp30to a valine in AdipoQ.

With the availability of the above-described expression vector it becamepossible to produce recombinant AdipoQ protein that could be used toconduct experiments in vitro and in vivo.

Example 4 describes the procedures that were used to prepare arecombinant form of the AdipoQ protein.

EXAMPLE 4 Production and Purification of Recombinant AdipoQ Protein

Bacteria containing the AdipoQ expression vector were cultured at 37° C.in LB medium under antibiotic selection until the OD₆₀₀ reached 0.2.Production of recombinant protein was then induced by addingisopropyl-β-D thiogalactopyranoside to a final concentration of 1 mM.Bacterial growth proceeded for an additional 16 hours at 37° C., afterwhich time the cultured bacteria were harvested by centrifugation.Bacteria were lysed according to standard laboratory procedures usinglysozyme in a buffer that included Tris HCl (pH 7.4), NaCl, PMSF andsodium deoxycholate. DNA in the crude lysate was degraded by sonication.After centrifugation to remove cellular debris, the recombinant proteinwas isolated from the cleared supernatant using a PROBOND column(Invitrogen, France). The nickel-charged resin of the column hasaffinity for the above-described hexahistidine peptide motif of therecombinant fusion protein. Elution was achieved in the presence ofimidazole. Following dialysis of the eluate, protein concentration wasmeasured by the standard Lowry method. Purity of the recombinant proteinwas verified by SDS polyacrylamide gel electrophoresis. A single band ofapparent molecular mass of about 33 kDa was observed on the protein gel.Notably, at this point the recombinant protein retained thehexahistidine protein domain.

We next employed an in vitro assay to investigate whether therecombinant AdipoQ, like C1q, stimulated LSR activity. Use of the invitro assay allowed us to particularly study different aspects ofLSR-mediated activity, including binding, internalization anddegradation of a model lipoprotein substrate.

Example 5 describes the methods that were used to prove that AdipoQstimulated LSR activity in vitro.

EXAMPLE 5 Recombinant AdipoQ Stimulates LSR Activity in CulturedHepatocytes

Primary cultures of rat hepatocytes were prepared and plated in 6-wellplates at 900,000 cells/well. After 48 hours the cells were washed oncewith 2 ml/well of phosphate buffered saline (PBS) and then incubated for30 minutes at 37° C. with 20 ng/ml of recombinant murine leptin.Thereafter the cells were further incubated 4 hours at 37° C. with 25μg/ml of recombinant AdipoQ and 20 μg/ml of ¹²⁵I-LDL in the presence orabsence of 0.6 mM oleat Binding, internalization and degradation of thelabeled LDL all were determined according to the above-referencedstandard methods.

The results presented in FIGS. 4A-C show that AdipoQ significantlyincreased the amount of LDL that was bound, internalized and degraded byhepatocytes. Indeed, the results particularly indicated that degradationof LDL was dramatically enhanced by AdipoQ treatment of the hepatocytes.It is worth noting that in this setting the increase of LSR activity dueto AdipoQ was measured in the presence of leptin. These resultsconfirmed that AdipoQ was capable of increasing LSR activity in primaryculture of rat hepatocytes.

Given the finding that AdipoQ dramatically enhanced LSR activity invitro, it was of interest to determine whether the same pattern ofactivity would be repeated in vivo. This possibility was tested byfeeding rats a high-fat meal, administering the rats with recombinantAdipoQ, measuring plasma triglycerides and comparing the results withmeasurements taken in rats that did not receive AdipoQ. As describedbelow, our findings indicated that administration with AdipoQdramatically reduced the level of plasma triglycerides following thehigh-fat test meal.

Example 6 describes the procedures that were used to demonstrate thatplasma triglyceride levels following a high-fat meal were reduced inanimals that had been injected with AdipoQ.

EXAMPLE 6 AdipoQ Reduces Postorandial Blood Livid Levels in Vivo

Overnight-fasted male Sprague-Dawley rats (400-450 g) were gavaged witha high-fat test meal (time=0) and immediately administered byintravenous injection into the femoral vein with either 300 μl of PBSalone or containing 1 mg of recombinant murine AdipoQ. The test mealconsisted of 60% fat (37% saturated, 27% mono, and 36% polyunsaturatedfatty acids), 20% protein and 20% carbohydrate, and provided 56 kcal ofenergy/kg of body weight. A second injection of AdipoQ was administered2 hours after the test meal. Blood samples were taken at two hourintervals and plasma triglyceride levels were determined by a standardenzymatic assay using reagents that had been purchased as a kit(Boehringer Mannheim).

The results presented in FIG. 5 show that AdipoQ substantially decreasedthe magnitude of the postprandial triglyceride response. Quantitativevalues presented in the Figure represent the mean±standard deviation (n3). Whereas the level of circulating triglycerides remainedsubstantially constant in the animals administered with AdipoQ, thelevel increased in control animals until reaching a peak at about 4hours. These in vivo results were consistent with the markedAdipoQ-dependent enhancement of LSR activity that we had observed invitro.

Example 7 describes the procedures used to demonstrate that AdipoQadministration promoted weight loss and reduction of plasma triglyceridelevels in normal animals. This was true even when the animals wereplaced on a high-fat diet. Notably, in this case the AdipoQ wasadministered by a slow infusion protocol instead of by injection.

EXAMPLE 7 Administration of AdipoQ by Infusion Stimulates Weight Lossand Reduction in Plasma Triglycerides

Osmotic pumps (Alzet) were surgically inserted into the abdominalcavities of 12 male 400-450 g Sprague-Dawley rats. The pumps containedeither 2 ml of PBS (pH 7.4) (control, n=6) or 2 ml mouse recombinantAdipoQ (5 mg/ml PBS, n=6). The pumps used in this procedure weredesigned to deliver 10 μl/hour (50 μg AdipoQ/hour). Animals were weighedand then housed individually in metabolic cages. Three animals in eachgroup were put either on regular chow diet or a high-fat diet ad libitum(day 0). The high-fat diet consisted of regular chow supplemented with2% (w/v) cholesterol, 10% (w/v) saturated fat in the form of vegetaline,10% (w/v) sunflower oil and 15% (w/v) sucrose. On day 3, the animalswere weighed and blood samples were obtained from the tail vein. Plasmatriglycerides were measured using an enzymatic kit.

The results presented in FIGS. 6A-B show that AdipoQ caused asignificant reduction in plasma triglyceride levels in test animals fedeither a regular or a high fat diet. Moreover, AdipoQ administrationcaused a reduction in body weight that was more pronounced in animalsfed the high fat diet.

Example 8 describes the procedures that defined yet another effect ofAdipoQ in vivo. More specifically, the results presented belowdemonstrate that test animals administered with AdipoQ unexpectedlyreduced their food intake.

EXAMPLE 8 AdipoQ Administration Promotes Reduction of Food Intake inGenetically Obese Mice

Both ob/ob and db/db mice housed in metabolic cages were injected dailyfor 5 days into the tail veins with either PBS alone or recombinantmurine AdipoQ (100 μg) dispersed in a PBS carrier. The amount of foodconsumed daily by each animal was monitored for the period of theexperiment.

The results presented in FIGS. 7A-B show that the average daily foodintake of obese mice was significantly reduced after AdipoQadministration. The graphic data reflect the average food intake andstandard deviation for 4 mice in each group, except for the db/dbcontrol group (n 3) in which one animal died before the end of theexperiment. Significantly, the AdipoQ-dependent reduction in food intakewas observed for the ob/ob and db/db groups of mice. This establishedthat AdipoQ was useful for controlling food intake in the absence ofleptin (ob/ob mice), and that AdipoQ was able to overcome the leptinresistance that is characteristic of db/db mice.

Example 9 describes a method that may be used to reduce plasmatriglycerides and body mass in humans. While this exemplary casedescribes a treatment of obese humans, it is to be understood thatnon-obese humans may also be administered with the medicament describedbelow. For purposes of procedures described in the following twoExamples, a population of individuals with body mass index >35 isrecruited and tested for diabetes (fasting plasma glucose levels >120mg/dl) and hypertriglyceridemia or “HTG” (fasting triglyceridelevels >150 mg/dl). Four groups of obese subjects are then constituted.These groups include: (1) subjects with obesity and no diabetes or HTG;(2) subjects with diabetes but no HTG; (3) subjects with HTG but nodiabetes; and (4) subjects with diabetes and HTG.

EXAMPLE 9 Administration of a Medicament that Includes AdipoQ

A population of obese human individuals is first identified and thenseparated into two random groups. The control group receives a dailyintravenous injection of a placebo for a period of from one week, twoweeks, one month or more than a month. The placebo comprises a 1.0 mlvolume of sterile PBS. Individuals in the treatment group receive anintravenous injection twice daily of a medicament that comprises 1.0 mlof sterile PBS containing recombinant AdipoQ at a dosage levelcorresponding to 2.5 mg AdipoQ/kg body mass. The recombinant AdipoQ isproduced according to good manufacturing procedures (GMP) in aprokaryotic expression system essentially according to the proceduresdescribed under Examples 3 and 4. Individuals in both groups consumehigh-fat meals, and serum triglyceride levels and body mass aremonitored regularly for the duration of the procedure.

At the end of the treatment period it is clear that individualsadministered with the medicament that included AdipoQ exhibitsubstantially reduced plasma triglyceride levels relative to the controlgroup. Moreover, there is evidence that these individuals haveexperienced measurable weight reduction.

Example 10 describes how ApM1 can be used to stimulate reduction inplasma triglyceride levels and body mass.

EXAMPLE 10 ApM1 Administration Reduces Plasma Triglycerides and BodyMass

A population of obese human individuals is first identified and thenseparated into two random groups. The control group receives a dailyintravenous injection of a placebo for a period of from one week, twoweeks, one month or more than a month. The placebo comprises a 1.0 mlvolume of sterile PBS. Individuals in the treatment group receive anintravenous injection twice daily of a medicament that comprises 1.0 mlof sterile PBS containing human ApM1 at a dosage level corresponding to2.5 mg ApM1/kg body mass. The human ApM1 is a recombinant materialproduced according to GMP standards. For this purpose a prokaryoticexpression system is used essentially according to the proceduresdescribed under Examples 3 and 4, except that human ApM1 cDNA issubstituted for the murine AdipoQ cDNA described in the Examples.Individuals in both groups consume high-fat meals. Serum triglyceridelevels and body mass are monitored for both groups of individuals.

At the end of the treatment period individuals in the group administeredwith the medicament that included ApM1 exhibit substantially reducedplasma triglyceride levels relative to the control group. Moreover,these individuals have experienced measurable weight reduction.

Another aspect of the present invention relates to the preparation of amedicament for influencing the partitioning of dietary lipids betweenthe adipose tissues and the liver in an individual. For example, themedicaments may increase or decrease the level of lipolysis which occursin the liver. In particular, the medicaments may increase or decreasethe level of lipolysis which occurs in the liver by increasing ordecreasing the activity of LSR.

Such medicaments can be used in procedures for reducing the amount ofdietary lipids stored in the adipose tissue or in procedures forincreasing the amount of dietary lipids stored in the adipose tissuedepending on the nature of the condition which is to be treated. Inparticular, such medicaments increase or decrease the activities ofcompounds (including AdipoQ, ApM1, C1q, any of the above-describedcompounds analogous to C1q, compounds having at least one consensussequence selected from the group consisting of SEQ ID NO:1 and SEQ IDNO:2, compounds comprising an amino acid sequence having at least 25%homology to a sequence selected from the group consisting of SEQ ID NOs.7-14, compounds comprising an amino acid sequence having at least 50%homology to a sequence selected from the group consisting of SEQ ID NOs.7-14, and compounds comprising an amino acid sequence having at least80% homology to a sequence selected from the group consisting of SEQ IDNOs. 7-14) which increase the amount of dietary lipids partitioned tothe liver.

Medicaments which increase the activity of these compounds in anindividual may be used to reduce food intake in obese individuals, toreduce the levels of free fatty acids in obese individuals, to decreasethe body weight of obese individuals, or to treat a variety of obesityrelated conditions. Such obesity related conditions includeatherosclerosis (which may result from elevated levels of free fattyacids and chylomicron remnants in the plasma), obesity-related insulinresistance resulting from fatty acids in the plasma or fatty acidsproduced by extracellular lipolysis (Walker, M., Metabolism 44: 18-20(1995); Lonnroth, P., Intern. Med. Suppl. 735: 23-29 (1991); Hannes, M.M. et al., Int. J. Obes. 14: 831-841 (1990), obesity-relatedhypertension resulting from fatty acids in the plasma or fatty acidsproduced by extracellular lipolysis (Goodfriend and Egan, J. Med.344:1649-1654 (1996), microangiopathic lesions resulting fromobesity-related Type II diabetes, and ocular and renal lesions caused bymicroangiopathy in obese subjects with Type II diabetes.

Medicaments which decrease the activity of compounds which increase thepartitioning of dietary lipids to the liver (including AdipoQ, ApM1,C1q, any of the above-described compounds analogous to C1q, compoundshaving at least one consensus sequence selected from the groupconsisting of SEQ ID NO:1 and SEQ ID NO:2, compounds comprising an aminoacid sequence having at least 25% homology to a sequence selected fromthe group consisting of SEQ ID NOs. 7-14, compounds comprising an aminoacid sequence having at least 50% homology to a sequence selected fromthe group consisting of SEQ ID NOs. 7-14, and compounds comprising anamino acid sequence having at least 80% homology to a sequence selectedfrom the group consisting of SEQ ID NOs. 7-14) may be used to treatconditions such as cachexia in subjects with neoplastic orpara-neoplastic syndrome or eating disorders.

A variety of techniques may be used to increase the activity ofcompounds which increase the partitioning of dietary lipids to theliver. In particular, the activity of these compounds may be increasedby directly administering these compounds (including AdipoQ, ApM1, C1q,any of the above-described compounds analogous to C1q, or compoundshaving at least one consensus sequence selected from the groupconsisting of SEQ ID NO:1 and SEQ ID NO:2, compounds comprising an aminoacid sequence having at least 25% homology to a sequence selected fromthe group consisting of SEQ ID NOs. 7-14, compounds comprising an aminoacid sequence having at least 50% homology to a sequence selected fromthe group consisting of SEQ ID NOs. 7-14, and compounds comprising anamino acid sequence having at least 80% homology to a sequence selectedfrom the group consisting of SEQ ID NOs.: 7-14 or a fragment of thepreceding compounds) to the individual in any of the pharmaceuticallyacceptable formulations described above. Routes of administration ofthese compounds, as well as appropriate doses of these agents, have alsobeen provided above.

Alternatively, the activity of compounds which increase the partitioningof dietary lipids to the liver may be increased by increasing theexpression of the genes encoding these compounds using gene therapy. Insuch procedures, a nucleic acid encoding a compound, or a portion of acompound, which increases the partitioning of dietary lipids to theliver (including AdipoQ, ApM1, C1q, any of the above-described compoundsanalogous to C1q, derivatives of any of the preceding compounds,compounds having at least one consensus sequence selected from the groupconsisting of SEQ ID NO:1 and SEQ ID NO:2, compounds comprising an aminoacid sequence having at least 25% homology to a sequence selected fromthe group consisting of SEQ ID NOs. 7-14, compounds comprising an aminoacid sequence having at least 50% homology to a sequence selected fromthe group consisting of SEQ ID NOs. 7-14, and compounds comprising anamino acid sequence having at least 80% homology to a sequence selectedfrom the group consisting of SEQ ID NOs. 7-14 or a fragment of thepreceding compounds) is transiently or stably introduced into theindividual to be treated.

The nucleic acid encoding a compound, or a portion of a compound, whichincreases the partitioning of dietary lipids to the liver (includingAdipoQ, ApM1, C1q, any of the above-described compounds analogous toC1q, or compounds having at least one consensus sequence selected fromthe group consisting of SEQ ID NO:1 and SEQ ID NO:2, compoundscomprising an amino acid sequence having at least 25% homology to asequence selected from the group consisting of SEQ ID NOs. 7-14,compounds comprising an amino acid sequence having at least 50% homologyto a sequence selected from the group consisting of SEQ ID NOs. 7-14,and compounds comprising an amino acid sequence having at least 80%homology to a sequence selected from the group consisting of SEQ IDNOs.: 7-14 or a fragment of the preceding compounds) is operably linkedto a promoter capable of directing its expression. The promoter may beany of the promoters familiar to those of skill in the art including theRous Sarcoma Virus promoter, the SV40 promoter, and the humancytomegalovirus promoter. In some embodiments, the promoter may be aliver-specific promoter. In further embodiments, the promoter may be thepromoter from the LSR gene.

Additional vectors and promoters suitable for use in gene therapyinclude the parvovirus vectors disclosed in U.S. Pat. No. 5,252,479, theadenovirus vectors disclosed in U.S. Pat. No. 5,585,362, and the Harveymurine sarcoma virus vectors disclosed in U.S. Pat. No. 5,166,059. Othergene therapy vectors familiar to those of skill in the art may also beused, including moloney murine leukemia virus vectors, pLJ, pZIP, pWeand pEM.

Alternatively, the nucleic acid encoding a compound, or a portion of acompound, which increases the partitioning of dietary lipids to theliver (including AdipoQ, ApM1, C1q, any of the above-described compoundsanalogous to C1q, or compounds having at least one consensus sequenceselected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2,compounds comprising an amino acid sequence having at least 25% homologyto a sequence selected from the group consisting of SEQ ID NOs. 7-14,compounds comprising an amino acid sequence having at least 50% homologyto a sequence selected from the group consisting of SEQ ID NOs. 7-14,and compounds comprising an amino acid sequence having at least 80%homology to a sequence selected from the group consisting of SEQ ID NOs.7-14 or a fragment of the preceding compounds) may be linked to apromoter capable of directing its expression and introduced into theindividual as naked DNA using procedures such as those described in U.S.Pat. No. 5,558,059.

In further approaches, a nucleic acid encoding AdipoQ, ApM1, C1q, any ofthe above-described compounds analogous to C1q, a compounds having atleast one consensus sequence selected from the group consisting of SEQID NO:1 and SEQ ID NO:2, a compound comprising an amino acid sequencehaving at least 25% homology to a sequence selected from the groupconsisting of SEQ ID NOs.: 7-14, a compound comprising an amino acidsequence having at least 50% homology to a sequence selected from thegroup consisting of SEQ ID NOs. 7-14, a compound comprising an aminoacid sequence having at least 80% homology to a sequence selected fromthe group consisting of SEQ ID NOs. 7-14 or a fragment of any of thepreceding compounds may be introduced into cells, such as fibroblastcells, using the gene therapy or naked DNA techniques described above.The cells, such as fibroblast cells, may be enclosed in a lattice ofcollagen and synthetic fibers coated with basic fibroblast growth factorso as to form an organoid. The organoid may then be transplanted into ahost animal. Techniques for producing organoids are disclosed in Bohl etal., Gene Ther. 2:197-202 (1995) and Descamps et al., Gene Ther.2:411-417 (1995).

In another approach, the genes (or portions thereof) which encodecompounds which increase the partitioning of dietary lipids to the liver(including AdipoQ, ApM1, C1q, any of the above-described compoundsanalogous to C1q, compounds having at least one consensus sequenceselected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2,compounds comprising an amino acid sequence having at least 25% homologyto a sequence selected from the group consisting of SEQ ID NOs. 7-14,compounds comprising an amino acid sequence having at least 50% homologyto a sequence selected from the group consisting of SEQ ID NOs. 7-14,and compounds comprising an amino acid sequence having at least 80%homology to a sequence selected from the group consisting of SEQ ID NOs.7-14) may be mutagenized to create derivative proteins or peptides whichhave a greater ability to increase the partitioning of dietary lipids tothe liver than the wild type proteins. A variety of mutagenesisprocedures are known to those of skill in the art, including sitedirected mutagenesis and random chemical mutagenesis. For example, anyof the mutagenesis procedures disclosed in Ausebel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons (1998) may be used.

The proteins or peptides encoded by the mutagenized genes are insertedinto expression vectors, isolated using techniques familiar to thoseskilled in the art, and tested to determine whether they have greateractivity than the wild type proteins using the procedures describedbelow.

Alternatively, rather than preparing derivatives using the mutagenesisprocedures described above, combinatorial chemistry techniques whichpermit the generation of a large number of derivative peptides in vitromay be used.

Derivative proteins or peptides having increased activity relative tothe wild type may be identified by comparing their activity to theactivity of the wild type proteins or peptides in the rat hepatocyteassay of Example 5. Those derivative proteins or peptides which haveincreased activity relative to the wild type proteins may then befurther analyzed in the postprandial lipemic response assay of Example6, the plasma triglyceride assay of Example 7, the food intake assay ofExample 8 or the weight loss assay of Example 7.

Those derivative proteins or peptides having increased activity relativeto the wild type proteins may be used in medicaments to increase thepartitioning of dietary lipids to the liver. In such medicaments, thederivative protein or peptide may be administered to the individual in apharmaceutically acceptable carrier such as those described above. Thederivative protein or peptide may be administered through any of theroutes and at any of the dosages described above.

In addition, as discussed above, small molecules, drugs, or othercompounds which increase the activity of a compound which increases thepartitioning of dietary lipids to the liver may be obtained by using avariety of synthetic approaches familiar to those skilled in the art,including combinatorial chemistry based techniques. Candidate smallmolecules, drugs, or other compounds may be evaluated by determiningtheir ability to increase the activity of a compound which increases thepartitioning of dietary lipids to the liver in the rat hepatocyte assayof Example 5. Those compounds which increase the activity of a compoundwhich increases the partitioning of dietary lipids to the liver in therat hepatocyte assay may be further evaluated in the postprandiallipemic response assay of Example 6, the plasma triglyceride assay ofExample 7, the food intake assay of Example 8, or the weight loss assayof Example 7.

As described above, the present invention also relates to medicamentsfor reducing the activity of compounds which increase the partitioningof dietary lipids to the liver. Such medicaments may be used to treatconditions such as those described above in which it is desirable todecrease the partitioning of dietary lipids to the liver (i.e. toincrease the partitioning of dietary lipids to the adipose tissue). Theactivity of compounds which increase the partitioning of dietary lipidsto the liver (including AdipoQ, ApM1, C1q, any of the above-describedcompounds analogous to C1q, or compounds having at least one consensussequence selected from the group consisting of SEQ ID NO:1 and SEQ IDNO:2, compounds comprising an amino acid sequence having at least 25%homology to a sequence selected from the group consisting of SEQ ID NOs.7-14, compounds comprising an amino acid sequence having at least 50%homology to a sequence selected from the group consisting of SEQ ID NOs.7-14, and compounds comprising an amino acid sequence having at least80% homology to a sequence selected from the group consisting of SEQ IDNOs. 7-14 or a fragment of the preceding compounds) may be reduced usinga variety of methods, including the methods described below.

The partitioning of dietary lipids to the liver may also be increased bypreparing an antibody which binds to the γ subunit, the C1q receptor(gC1q-R) or a protein related thereto, as well as fragments of theseproteins. Such antibodies may modulate the interaction between LSR andthe γ subunit, the C1q receptor (gC1q-R) or a protein related thereto ina manner which increases the partitioning of dietary lipids to theliver. The antibodies may be any of the antibodies described below.

In one procedure for reducing the activity of a compound which increasesthe partitioning of dietary lipids to the liver, an antibody whichinhibits the activity of the compound is administered to an individual.The antibody may be polygonal or monoclonal.

Polyclonal antibodies capable of specifically binding to a compoundwhich increases the partitioning of dietary lipids to the liver may beobtained by using the compound (or a fragment thereof) as an immunogenin the procedures described in Example 1 above. Alternatively,polyclonal antibodies may be generated against the γ subunit, the C1qreceptor (gC1q-R) or a protein related thereto, as well as fragments ofthese proteins.

Monoclonal antibodies to compounds which increase the partitioning ofdietary lipids to the liver can be prepared from murine hybridomasaccording to the classical method of Kohler, G. and Milstein, C., Nature256:495 (1975) or derivative methods thereof. Briefly, a mouse isrepetitively inoculated with a few micrograms of the compound or afragment thereof (such as AdipoQ, ApM1, C1q, any of the above-describedcompounds analogous to C1q, or compounds having at least one consensussequence selected from the group consisting of SEQ ID NO:1 and SEQ IDNO:2, compounds comprising an amino acid sequence having at least 25%homology to a sequence selected from the group consisting of SEQ IDNOs.: 7-14, compounds comprising an amino acid sequence having at least50% homology to a sequence selected from the group consisting of SEQ IDNOs.: 7-14, and compounds comprising an amino acid sequence having atleast 80% homology to a sequence selected from the group consisting ofSEQ ID NOs.: 7-14 or a fragment of the preceding compounds) over aperiod of a few weeks. Alternatively, monoclonal antibodies may begenerated against the γ subunit, the C1q receptor (gC1q-R) or a proteinrelated thereto, as well as fragments of these proteins.

The mouse is then sacrificed, and the antibody producing cells of thespleen isolated. The spleen cells are fused by means of polyethyleneglycol with mouse myeloma cells, and the excess unfused cells destroyedby growth of the system on selective media comprising aminopterin (HATmedia). The successfully fused cells are diluted and aliquots of thedilution placed in wells of a microtiter plate where growth of theculture is continued. Antibody-producing clones are identified bydetection of antibody in the supernatant fluid of the wells byimmunoassay procedures, such as Elisa, as originally described byEngvall, E., Meth. Enzymol. 70:419 (1980), and derivative methodsthereof. Selected positive clones can be expanded and their monoclonalantibody product harvested for use. Detailed procedures for monoclonalantibody production are described in Davis, L. et al. Basic Methods inMolecular Biology Elsevier, N.Y. Section 21-2.

Antibodies which are capable of inhibiting the activity of compoundswhich increase the partitioning of dietary lipids to the liver(including AdipoQ, ApM1, C1q, any of the above-described compoundsanalogous to C1q compounds having at least one consensus sequenceselected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2,compounds comprising an amino acid sequence having at least 25% homologyto a sequence selected from the group consisting of SEQ ID NOs.: 7-14,compounds comprising an amino acid sequence having at least 50% homologyto a sequence selected from the group consisting of SEQ ID NOs. 7-14,and compounds comprising an amino acid sequence having at least 80%homology to a sequence selected from the group consisting of SEQ IDNOs.: 7-14) may be identified by contacting the compound with increasingamounts of the monoclonal or polyclonal antibodies prior to conductingor while conducting the assay described in Example 5 with the compound.Those antibodies which reduce binding, internalization, and/ordegradation in the rat hepatocyte assay may be tested for in vivoactivity by administering increasing amounts of the antibodies to miceand determining the ability of the antibodies to inhibit thecompound-mediated reduction in postprandial triglyceride response in theassay described in Example 6 above, the ability of the antibodies toinhibit the compound-mediated reduction in plasma triglycerides in theassay described in Example 7 above, the ability of the antibodies toinhibit the compound-mediated reduction of food intake in obese mice inthe assay described in Example 8 above, or the ability of the antibodiesto inhibit the compound-mediated weight loss in the assay described inExample 7.

The partitioning of dietary lipids to the liver may also be reduced bypreparing an antibody which binds to the γ subunit, the C1q receptor(gC1q-R) or a protein related thereto, as well as fragments of theseproteins. Such antibodies may modulate the interaction between AdipoQ,ApM1, or analogous proteins and the γ subunit, the C1q receptor (gC1q-R)or a protein related thereto in a manner which reduces the partitioningof dietary lipids to the liver. The antibodies may be any of theantibodies described above.

Alternatively, the partitioning of dietary lipids to the liver may alsobe reduced using fragments of antibodies which retain the ability tospecifically bind AdipoQ, ApM1, C1q, any of the above-describedcompounds analogous to C1q, compounds having at least one consensussequence selected from the group consisting of SEQ ID NO:1 and SEQ IDNO:2, compounds comprising an amino acid sequence having at least 25%homology to a sequence selected from the group consisting of SEQ IDNOs.: 7-14, compounds comprising an amino acid sequence having at least50% homology to a sequence selected from the group consisting of SEQ IDNOs. 7-14, compounds comprising an amino acid sequence having at least80% homology to a sequence selected from the group consisting of SEQ IDNOs. 7-14, the γ subunit, the C1q receptor (gC1q-R) or a protein relatedthereto, as well as fragments of these proteins. For example, thefragments may be Fab fragments, which may be prepared using methodsfamiliar to those of skill in the art.

Alternatively, the antibodies may comprise humanized antibodies orsingle chain antibodies. A variety of methods for making humanizedantibodies or single chain antibodies are familiar to those skilled inthe art, including the techniques described in U.S. Pat. Nos. 5,705,154,5,565,332, and 5,608,039.

Those antibodies which inhibit the compound-mediated effects in one ormore of the assays described above may then be used in medicaments forreducing the activity of compounds which increase the partitioning ofdietary lipids to the liver. The antibodies may be administered toindividuals in a pharmaceutically acceptable carrier such as thosedescribed above.

Alternatively, the activity of compounds which increase the partitioningof dietary lipids to the liver (including AdipoQ, ApM1, C1q, any of theabove-described compounds analogous to C1q, or compounds having at leastone consensus sequence selected from the group consisting of SEQ ID NO:1and SEQ ID NO:2, compounds comprising an amino acid sequence having atleast 25% homology to a sequence selected from the group consisting ofSEQ ID NOs. 7-14, compounds comprising an amino acid sequence having atleast 50% homology to a sequence selected from the group consisting ofSEQ ID NOs. 7-14, and compounds comprising an amino acid sequence havingat least 80% homology to a sequence selected from the group consistingof SEQ ID NOs. 7-14, or a fragment of the preceding compounds) may bereduced by reducing the expression of the genes encoding the compounds.A variety of approaches may be used to reduce gene expression, includingantisense or triple helix based strategies.

In antisense approaches, nucleic acid sequences complementary to themRNA encoding the compound capable of increasing the partitioning ofdietary lipids to the liver are hybridized to the mRNA intracellularly,thereby blocking the expression of the protein encoded by the mRNA. Theantisense sequences may prevent gene expression through a variety ofmechanisms. For example, the antisense sequences may inhibit the abilityof ribosomes to translate the mRNA. Alternatively, the antisensesequences may block transport of the mRNA from the nucleus to thecytoplasm, thereby limiting the amount of mRNA available fortranslation. Another mechanism through which antisense sequences mayinhibit gene expression is by interfering with mRNA splicing. In yetanother strategy, the antisense nucleic acid may be incorporated in aribozyme capable of specifically cleaving the target mRNA.

The antisense nucleic acid molecules to be used in gene therapy may beeither DNA or RNA sequences. They may comprise a sequence complementaryto the sequence of a gene, or a portion of a gene, encoding a compoundwhich increases the partitioning of dietary lipids to the liver. Theantisense nucleic acids should have a length and melting temperaturesufficient to permit formation of an intracellular duplex havingsufficient stability to inhibit the expression of the mRNA in theduplex. Strategies for designing antisense nucleic acids suitable foruse in gene therapy are disclosed in Green et al., Ann. Rev. Biochem.55:569-597 (1986) and Izant and Weintraub, Cell 36:1007-1015 (1984),which are hereby incorporated by reference.

In some strategies, antisense molecules are obtained from a nucleotidesequence encoding a compound which increases the partitioning of dietarylipids to the liver by reversing the orientation of the coding regionwith respect to a promoter so as to transcribe the opposite strand fromthat which is normally transcribed in the cell. The antisense moleculesmay be transcribed using in vitro transcription systems such as thosewhich employ T7 or SP6 polymerase to generate the transcript. Anotherapproach involves transcription of the antisense nucleic acids in vivoby operably linking DNA containing the antisense sequence to a promoterin an expression vector.

Alternatively, oligonucleotides which are complementary to the strandnormally transcribed in the cell may be synthesized in vitro. Forexample, the oligonucleotides used in antisense procedures may beprepared on an oligonucleotide synthesizer or they may be purchasedcommercially from a company specializing in custom oligonucleotidesynthesis, such as GENSET, Paris, France.

The antisense nucleic acids are complementary to the corresponding mRNAand are capable of hybridizing to the mRNA to create a duplex. In someembodiments, the antisense sequences may contain modified sugarphosphate backbones to increase stability and make them less sensitiveto RNase activity. Examples of modifications suitable for use inantisense strategies are described by Rossi et al., Pharmacol. Ther.50(2):245-254, (1991).

Various types of antisense oligonucleotides complementary to genesencoding compounds which influence the partitioning of dietary lipids tothe liver (including genes encoding AdipoQ, ApM1, C1q, any of theabove-described compounds analogous to C1q, compounds having at leastone consensus sequence selected from the group consisting of SEQ ID NO:1and SEQ ID NO:2, compounds comprising an amino acid sequence having atleast 25% homology to a sequence selected from the group consisting ofSEQ ID NOs. 7-14, compounds comprising an amino acid sequence having atleast 50% homology to a sequence selected from the group consisting ofSEQ ID NOs.: 7-14, or compounds comprising an amino acid sequence havingat least 80% homology to a sequence selected from the group consistingof SEQ ID NOs.: 7-14) may be used. In one preferred embodiment, stableand semi-stable antisense oligonucleotides described in InternationalApplication No. PCT WO94/23026, hereby incorporated by reference, areused. In these molecules, the 3′ end or both the 3′ and 5′ ends areengaged in intramolecular hydrogen bonding between complementary basepairs. These molecules are better able to withstand exonuclease attacksand exhibit increased stability compared to conventional antisenseoligonucleotides.

In another preferred embodiment, the antisense oligodeoxynucleotidesdescribed in International Application No. WO 95/04141, the disclosureof which is incorporated herein by reference, are used.

In yet another preferred embodiment, the covalently cross-linkedantisense oligonucleotides described in International Application No. WO96/31523, hereby incorporated by reference, are used. These double- orsingle-stranded oligonucleotides comprise one or more, respectively,inter- or intra-oligonucleotide covalent cross-linkages, wherein thelinkage consists of an amide bond between a primary amine group of onestrand and a carboxyl group of the other strand or of the same strand,respectively, the primary amine group being directly substituted in the2′ position of the strand nucleotide monosaccharide ring, and thecarboxyl group being carried by an aliphatic spacer group substituted ona nucleotide or nucleotide analog of the other strand or the samestrand, respectively.

The antisense oligodeoxynucleotides and oligonucleotides disclosed inInternational Application No. WO 92/18522, incorporated by reference,may also be used. These molecules are stable to degradation and containat least one transcription control recognition sequence which binds tocontrol proteins and are effective as decoys therefor. These moleculesmay contain “hairpin” structures, “dumbbell” structures, “modifieddumbbell” structures, “cross-linked” decoy structures and “loop”structures.

In another preferred embodiment, the cyclic double-strandedoligonucleotides described in European Patent Application No. 0 572 287A2, hereby incorporated by reference are used. These ligatedoligonucleotide “dumbbells” contain the binding site for a transcriptionfactor and inhibit expression of the gene under control of thetranscription factor by sequestering the factor.

Use of the closed antisense oligonucleotides disclosed in InternationalApplication No. WO 92/19732, hereby incorporated by reference, is alsocontemplated. Because these molecules have no free ends, they are moreresistant to degradation by exonucleases than are conventionaloligonucleotides. These oligonucleotides may be multifunctional,interacting with several regions which are not adjacent to the targetmRNA.

It is further contemplated that the antisense oligonucleotide sequenceis incorporated into a ribozyme sequence to enable the antisense tospecifically bind and cleave its target mRNA. For technical applicationsof ribozyme and antisense oligonucleotides see Rossi et al., supra.

The appropriate level of antisense nucleic acids required to inhibitgene expression may be determined using in vitro expression analysis.The antisense molecule may be introduced into cells which express thetarget gene by diffusion, injection, infection or transfection usingprocedures known in the art. For example, if the target gene is theAdipoQ gene or a gene encoding an analogous protein (such as the AdipoQgene or the ApM1 gene), the antisense molecule may be introduced intoadipocytes.

The antisense molecules are introduced onto cell samples at a number ofdifferent concentrations preferably between 1×10⁻¹⁰M to 1×10⁻⁴M. Oncethe minimum concentration that can adequately control gene expression isidentified, the optimized dose is translated into a dosage suitable foruse in vivo. For example, an inhibiting concentration in culture of1×10⁻⁷ M translates into a dose of approximately 0.6 mg/kg bodyweight.Levels of oligonucleotide approaching 100 mg/kg body weight or highermay be possible after testing the toxicity of the oligonucleotide inlaboratory animals.

When using the antisense molecules as a medicament, the antisensenucleic acids can be introduced into the body of an individual to betreated as a bare or naked oligonucleotide, oligonucleotide encapsulatedin lipid, oligonucleotide sequence encapsidated by viral protein, or asan oligonucleotide operably linked to a promoter contained in anexpression vector. The expression vector may be any of a variety ofexpression vectors known in the art, including retroviral or viralvectors such as those described above, vectors capable ofextrachromosomal replication, or integrating vectors. The vectors may beDNA or RNA. It is additionally contemplated that cells from thevertebrate are removed, treated with the antisense oligonucleotide, andreintroduced into the vertebrate.

Alternatively, the activity of a compound which increases thepartitioning of dietary lipids to the liver may be reduced usingstrategies based on intracellular triple helix formation. Triple helixoligonucleotides are used to inhibit transcription from a genome. Theyare particularly useful for studying alterations in cell activity as itis associated with a particular gene. The gene encoding a compound whichincreases the partitioning of dietary lipids to the liver (such as thegene encoding AdipoQ, ApM1, C1q, any of the above-described compoundsanalogous to C1q or compounds having at least one consensus sequenceselected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2,compounds comprising an amino acid sequence having at least 25% homologyto a sequence selected from the group consisting of SEQ ID NOs. 7-14,compounds comprising an amino acid sequence having at least 50% homologyto a sequence selected from the group consisting of SEQ ID NOs.: 7-14,or compounds comprising an amino acid sequence having at least 80%homology to a sequence selected from the group consisting of SEQ ID NOs.7-14) more preferably, a portion of such a gene, can be used in triplehelix based approaches to inhibit gene expression. Traditionally,homopurine sequences were considered the most useful for triple helixstrategies. However, homopyrimidine sequences can also inhibit geneexpression. Such homopyrimidine oligonucleotides bind to the majorgroove at homopurine:homopyrimidine sequences. Thus, both types ofsequences from genes encoding compounds which increase the partitioningof dietary lipids to the liver may be used in triple helix basedapproaches such as the following.

The sequence of a gene encoding a compound which increases thepartitioning of dietary lipids to the liver is scanned to identify10-mer to 20-mer homopyrimidine or homopurine stretches which could beused in triple-helix based strategies for inhibiting gene expression. Insome embodiments, the natural (beta) anomers of the oligonucleotideunits can be replaced with alpha anomers to render the oligonucleotidemore resistant to nucleases. Further, an intercalating agent such asethidium bromide, or the like, can be attached to the 3′ end of thealpha oligonucleotide to stabilize the triple helix. For information onthe generation of oligonucleotides suitable for triple helix formationsee Griffin et al. (Science 245:967-971 (1989), which is herebyincorporated by this reference).

Following identification of candidate homopyrimidine or homopurinestretches, their efficiency in inhibiting gene expression may beassessed by introducing varying amounts of oligonucleotides containingthe candidate sequences into cells which normally express the targetgene and measuring the ability of the triple helix-forming nucleic acidsto inhibit gene expression. For example, if the target gene is theAdipoQ gene or a gene encoding an analogous protein such as ApM1, thetriple helix-forming nucleic acids may be introduced into adipocytes.

The oligonucleotides may be introduced into the cells using a variety ofmethods known to those skilled in the art, including but not limited tocalcium phosphate precipitation, DEAE-Dextran, electroporation,liposome-mediated transfection or native uptake. Treated cells aremonitored for reduced gene expression using techniques such as Northernblotting, RNase protection assays, or PCR based strategies to monitorthe transcription levels of the target gene in cells which have beentreated with the oligonucleotide.

The triple helix-forming oligonucleotides which are effective ininhibiting gene expression in tissue culture cells may then be used inmedicaments for reducing the activity of the compound which increasesthe partitioning of dietary lipids to the liver. The triplehelix-forming oligonucleotides may be introduced into an individualusing the techniques provided in the above description of antisensestrategies.

Alternatively, the activity of a compound which increases thepartitioning of dietary lipids to the liver may be reduced usingderivatives of the compound which inhibit its activity. For examplederivatives of compounds which increase the partitioning of dietarylipids to the liver (such as derivatives of AdipoQ, ApM1, C1q, any ofthe above-described compounds analogous to C1q or compounds having atleast one consensus sequence selected from the group consisting of SEQID NO:1 and SEQ ID NO:2, compounds comprising an amino acid sequencehaving at least 25% homology to a sequence selected from the groupconsisting of SEQ ID NOs.: 7-14, compounds comprising an amino acidsequence having at least 50% homology to a sequence selected from thegroup consisting of SEQ ID NOs.: 7-14, compounds comprising an aminoacid sequence having at least 80% homology to a sequence selected fromthe group consisting of SEQ ID NOs.: 7-14, or fragments of any of theseproteins) may be prepared using the mutagenesis or combinatorialchemistry procedures described above in connection with the preparationof derivatives having enhanced activity relative to the wild type.

Derivative proteins or peptides produced using the above procedures maybe used in medicaments for reducing the activity of compounds whichincrease the partitioning of dietary lipids to the liver. Such mutantproteins or peptides may reduce the activity of a compound whichincreases the partitioning of dietary lipids to the liver (includingAdipoQ, ApM1, C1q, any of the above-described compounds analogous toC1q, or compounds having at least one consensus sequence selected fromthe group consisting of SEQ ID NO:1 and SEQ ID NO:2, compoundscomprising an amino acid sequence having at least 25% homology to asequence selected from the group consisting of SEQ ID NOs. 7-14,compounds comprising an amino acid sequence having at least 50% homologyto a sequence selected from the group consisting of SEQ ID NOs.: 7-14,and compounds comprising an amino acid sequence having at least 80%homology to a sequence selected from the group consisting of SEQ IDNOs.: 7-14) through a variety of mechanisms, including by acting asantagonists to binding of the wild type proteins to their ligands. Forexample, the antagonists may have a reduced activity and can thus reducethe activity of the compound by competing with it.

Derivative proteins or peptides which are capable of inhibiting theactivity of the wild type protein may be identified by determining theirability to block the activity of the wild type proteins in assays suchas the rat hepatocyte assay of Example 5, the postprandial lipemicresponse assay of Example 6, the plasma triglyceride assay of Example 7,the food intake assay of Example 8, or the weight loss assay of Example7. Alternatively, the derivative proteins or peptides may be evaluatedby determining their ability to increase food intake or cause a weightgain when administered in the assays of Examples 7 and 8.

Those derivative proteins or peptides which inhibit the activity of thewild type proteins may be used in medicaments for reducing the activityof a compound which increases the partitioning of dietary lipids to theliver. In such medicaments, the derivative protein or peptide may beadministered to the individual in a pharmaceutically acceptable carriersuch as those described above. The derivative protein or peptide may beadministered through any of the routes and at any of the dosagesdescribed above.

In addition, as discussed above, small molecules, drugs, or othercompounds which reduce the activity of a compound which increases thepartitioning of dietary lipids to the liver may be obtained by using avariety of synthetic approaches familiar to those skilled in the art,including combinatorial chemistry based techniques. Candidate smallmolecules, drugs, or other compounds may be evaluated by determiningtheir ability to inhibit the activity of a compound which increases thepartitioning of dietary lipids to the liver in the rat hepatocyte assayof Example 5. Those compounds which inhibit the activity of a compoundwhich increases the partitioning of dietary lipids to the liver in therat hepatocyte assay may be further evaluated in the postprandiallipemic response assay of Example 6, the plasma triglyceride assay ofExample 7, the food intake assay of Example 8, or the weight loss assayof Example 8.

Thus, one aspect of the present invention is an agent which increasesthe activity of a compound which increases the partitioning of dietarylipids to the liver for use as a pharmaceutical. In particular, theagent may be used for treating a condition selected from the groupconsisting of obesity, obesity-related atherosclerosis, obesity-relatedinsulin resistance, obesity-related hypertension, obesity-relatedmicroangiopathic lesions, obesity-related ocular lesions,obesity-related renal lesions, and other conditions in which it isdesirable to increase the partitioning of dietary lipids to the liver.In particular, the preceding conditions may be treated by administeringa therapeutically effective amount of a compound which increases thepartitioning of dietary lipids to the liver in a pharmaceuticallyacceptable carrier to an individual suffering from the precedingconditions. In some embodiments the medicament can be administered to anindividual who has been determined to have less than the normal level ofactivity of a compound which increases the partitioning of dietarylipids to the liver. In particular, the medicament may comprise AdipoQ,ApM1, C1q, any of the above-described compounds analogous to C1q orcompounds having at least one consensus sequence selected from the groupconsisting of SEQ ID NO:1 and SEQ ID NO:2, compounds comprising an aminoacid sequence having at least 25% homology to a sequence selected fromthe group consisting of SEQ ID NOs.: 7-14, compounds comprising an aminoacid sequence having at least 50% homology to a sequence selected fromthe group consisting of SEQ ID NOs.: 7-14, compounds comprising an aminoacid sequence having at least 80% homology to a sequence selected fromthe group consisting of SEQ ID NOs.: 7-14, or fragments of any of theseproteins. Alternatively, the medicament may comprise a derivative of thepreceding compounds which exhibits greater activity than the wild typecompound or a nucleic acid which increases the level of expression ofthe preceding compounds in the individual.

Another aspect of the present invention is an agent which inhibits theactivity of a compound wich increases the partitioning of dietary lipidsto the liver for use as a pharmaceutical. The pharmaceutical may be usedfor treating a condition selected from the group consisting of cachexiain subjects with neoplastic or para-neoplastic syndrome, eatingdisorders, and other conditions in which it is desirable to reduce thepartitioning of dietary lipids to the liver. In particular, thepreceding conditions may be treated by administering a therapeuticallyeffective amount of an agent which inhibits the activity of a compoundwhich increases the partitioning of dietary lipids to the liver in apharmaceutically acceptable carrier to an individual suffering from thepreceding conditions. In some embodiments the medicament can beadministered to an individual who has been determined to have more thanthe normal level of activity of a compound which increases thepartitioning of dietary lipids to the liver. In particular, themedicament may comprise an agent which inhibits the activity of AdipoQ,ApM1, C1q, any of the above-described compounds analogous to C1q, acompound having at least one consensus sequence selected from the groupconsisting of SEQ ID NO:1 and SEQ ID NO:2, compounds comprising an aminoacid sequence having at least 25% homology to a sequence selected fromthe group consisting of SEQ ID NOs.: 7-14, compounds comprising an aminoacid sequence having at least 50% homology to a sequence selected fromthe group consisting of SEQ ID NOs.: 7-14, or a compound comprising anamino acid sequence having at least 80% homology to a sequence selectedfrom the group consisting of SEQ ID NOs.: 7-14. For example themedicament may comprise an antibody which inhibits the activity ofAdipoQ, ApM1, C1q, any of the above-described compounds analogous toC1q, a compound having at least one consensus sequence selected from thegroup consisting of SEQ ID NO:1 and SEQ ID NO:2, a compound comprisingan amino acid sequence having at least 25% homology to a sequenceselected from the group consisting of SEQ ID NOs.: 7-14, a compoundcomprising an amino acid sequence having at least 50% homology to asequence selected from the group consisting of SEQ ID NOs.: 7-14, or acompound comprising an amino acid sequence having at least 80% homologyto a sequence selected from the group consisting of SEQ ID NOs.: 7-14.The medicament may also comprise a derivative of AdipoQ, ApM1, C1q, anyof the above-described compounds analogous to C1q, a compounds having atleast one consensus sequence selected from the group consisting of SEQID NO:1 and SEQ ID NO:2, a compound comprising an amino acid sequencehaving at least 25% homology to a sequence selected from the groupconsisting of SEQ ID NOs.: 7-14, a compound comprising an amino acidsequence having at least 50% homology to a sequence selected from thegroup consisting of SEQ ID NOs. 7-14, and compounds comprising an aminoacid sequence having at least 80% homology to a sequence selected fromthe group consisting of SEQ ID NOs.: 7-14, or a fragment of thepreceding compounds which inhibits the activity of AdipoQ, ApM1, C1q,any of the above-described compounds analogous to C1q, a compound havingat least one consensus sequence selected from the group consisting ofSEQ ID NO:1 and SEQ ID NO:2, a compound comprising an amino acidsequence having at least 25% homology to a sequence selected from thegroup consisting of SEQ ID NOs.: 7-14, a compound comprising an aminoacid sequence having at least 50% homology to a sequence selected fromthe group consisting of SEQ ID NOs. 7-14, or a compound comprising anamino acid sequence having at least 80% homology to a sequence selectedfrom the group consisting of SEQ ID NOs. 7-14. Alternatively, themedicament may comprise a nucleic acid, such as an antisense nucleicacid or a triple helix-forming nucleic acid, which alters the expressionor decreases the level of expression of AdipoQ, ApM1, C1q, any of theabove-described compounds analogous to C1q, a compound having at leastone consensus sequence selected from the group consisting of SEQ ID NO:1and SEQ ID NO:2, a compound comprising an amino acid sequence having atleast 25% homology to a sequence selected from the group consisting ofSEQ ID NOs.: 7-14, a compound comprising an amino acid sequence havingat least 50% homology to a sequence selected from the group consistingof SEQ ID NOs.: 7-14, or a compound comprising an amino acid sequencehaving at least 80% homology to a sequence selected from the groupconsisting of SEQ ID NOs.: 7-14 in the individual.

Obese individuals express lower than normal levels of AdipoQ or AdipoQrelated compounds. (Hu et al., J. Biol. Chem. 271:10697-10703 (1996)).Obese individuals having decreased activity of AdipoQ, ApM1, oranalogous compounds in their plasma, body fluids, or body tissues may beat risk of developing a variety of conditions associated withpartitioning lower than normal levels of dietary lipids to the liver(i.e. partitioning higher than normal levels of dietary lipids to theadipose tissues). In particular, such individuals may suffer fromobesity-related atherosclerosis, obesity-related insulin resistance,obesity-related hypertension, obesity-related microangiopathic lesions,obesity-related ocular lesions, and obesity-related renal lesions.Accordingly, another aspect of the present invention is a method fordetermining whether an obese individual is at risk of suffering from acondition selected from the group consisting of obesity-relatedatherosclerosis, obesity-related insulin resistance, obesity-relatedhypertension, obesity-related microangiopathic lesions, obesity-relatedocular lesions, obesity-related renal lesions comprising determiningwhether the individual has a below normal level of activity of AdipoQ,ApM1 or analogous compounds in plasma, body fluids, or body tissues.

The level of AdipoQ, ApM1 or analogous compounds in plasma, body fluids,or body tissues may be determined using a variety approaches. Inparticular, the level may be determined using ELISA, Western Blots, orprotein electrophoresis.

Another aspect of the present invention relates to methods ofidentifying molecules which bind to the γ subunit. As discussed above,the γ subunit may be the C1q receptor (gC1q-R) or a protein relatedthereto. Accordingly, as used below, the terminology “γ subunit” willrefer to gC1q-R or the related protein which makes up the γ subunit ofthe LSR complex.

Molecules which bind to the γ subunit may be used in the medicaments andmethods of the present invention to increase or decrease thepartitioning of dietary lipids to the liver. For example, such moleculesmay act as agonists or antagonists to stimulate or decrease the activityof LSR.

There are numerous methods available for identifying γ subunit ligands.One such method is described in U.S. Pat. No. 5,270,170, the disclosureof which is incorporated herein by reference. Briefly, in this method, arandom peptide library is constructed. The random peptide librarycomprises a plurality of vectors encoding fusions between peptides to betested for γ subunit binding activity and a DNA binding protein, such asthe lac repressor encoded by the lacI gene. The vectors in the randompeptide library also contain binding sites for the DNA binding protein,such as the lacO site in the case where the DNA binding protein is thelac repressor. The random peptide library is introduced into a hostcell, where the fusion protein is expressed. The host cells are thenlysed under conditions which permit the DNA binding portion of thefusion protein to bind to the DNA binding sites on the vector.

The vectors having the fusion proteins bound thereto are placed incontact with immobilized γ subunit, or an immobilized fragment of γsubunit under conditions which permit peptides to bind specifically. Forexample, γ subunit or a fragment thereof may be immobilized by affixingit to a surface such as a plastic plate or a particle. In particular,the immobilized fragment of γ subunit may comprise the C1q, AdipoQ orApM1 binding site.

Those vectors which encode random peptides capable of binding to theimmobilized γ subunit, or a fragment thereof, or the C1q, AdipoQ or ApM1binding site thereof will be specifically retained on the surface viathe interaction between the peptide and γ subunit, a fragment of the γsubunit, or the C1q, AdipoQ or ApM1 binding site thereof.

Alternatively, molecules capable of binding to the γ subunit may beidentified using two-hybrid systems such as the Matchmaker Two HybridSystem 2 (Catalog No. K1604-1, Clontech). As described in the manualaccompanying the Matchmaker Two Hybrid System 2 (Catalog No. K1604-1,Clontech), which is incorporated herein by reference, nucleic acidsencoding the γ subunit, a fragment thereof, or a fragment comprising theC1q, AdipoQ or ApM1 binding site are inserted into an expression vectorsuch that they are in frame with DNA encoding the DNA binding domain ofthe yeast transcriptional activator GAL4. Nucleic acids in a librarywhich encode proteins or peptides which might interact with the γsubunit, a fragment of the γ subunit, or the C1q, AdipoQ or ApM1 bindingsite are inserted into a second expression vector such that they are inframe with DNA encoding the activation domain of GAL4. The twoexpression plasmids are transformed into yeast and the yeast are platedon selection medium which selects for expression of selectable markerson each of the expression vectors as well as GAL4 dependent expressionof the HIS3 gene. Transformants capable of growing on medium lackinghistidine are screened for GAL4 dependent lacZ expression. Those cellswhich are positive in both the histidine selection and the lacZ assaycontain plasmids encoding proteins or peptides which interact with the γsubunit, a fragment thereof, or the C1q, AdipoQ or ApM1 binding site.

Alternatively, to study the interaction of the γ subunit, a fragmentthereof, or a fragment comprising the C1q, AdipoQ or ApM1 binding sitethereof with drugs or small molecules, such as molecules generatedthrough combinatorial chemistry approaches, the microdialysis coupled toHPLC method described by Wang et al., Chromatographia, 44, 205-208(1997)or the affinity capillary electrophoresis method described by Busch etal., J. Chromatogr. 777:311-328 (1997), the disclosures of which areincorporated herein by reference can be used.

In further methods, proteins, peptides, drugs, small molecules, or othercompounds which interact with the γ subunit, a fragment thereof, or afragment comprising the C1q, AdipoQ or ApM1 binding site thereof may beidentified using assays such as the following. The molecule to be testedfor binding is labeled with a detectable label, such as a fluorescent,radioactive, or enzymatic tag and placed in contact with immobilized γsubunit, a fragment thereof, or a fragment comprising the C1q, AdipoQ orApM1 binding site thereof under conditions which permit specific bindingto occur. After removal of non-specifically bound molecules, boundmolecules are detected using appropriate means.

Alternatively, proteins, peptides, drugs, small molecules, or othercompounds which bind to γ subunit, a fragment thereof, or a fragmentcomprising the C1q, AdipoQ or ApM1 binding site thereof may beidentified using competition experiments. In such assays, the γ subunit,a fragment thereof, or a fragment comprising the C1q, AdipoQ or ApM1binding site thereof is immobilized to a surface, such as a plasticplate. Increasing amounts of the proteins, peptides, drugs, smallmolecules, or other compounds are placed in contact with the immobilizedγ subunit, a fragment thereof, or a fragment comprising the C1q, AdipoQor ApM1 binding site thereof in the presence of a detectably labeledknown γ subunit ligand, such as AdipoQ, C1q, any of the above-describedcompounds analogous to C1q, a compound having at least one consensussequence selected from the group consisting of SEQ ID NO:1 and SEQ IDNO:2, a compound comprising an amino acid sequence having at least 25%homology to a sequence selected from the group consisting of SEQ IDNOs.: 7-14, a compound comprising an amino acid sequence having at least50% homology to a sequence selected from the group consisting of SEQ IDNOs.: 7-14, or a compound comprising an amino acid sequence having atleast 80% homology to a sequence selected from the group consisting ofSEQ ID NOs.: 7-14. For example, the γ subunit ligand may be detectablylabeled with a fluorescent, radioactive, or enzymatic tag. The abilityof the test molecule to bind the γ subunit, a fragment thereof, or afragment comprising the C1q, AdipoQ or ApM1 binding site thereof isdetermined by measuring the amount of detectably labeled known ligandbound in the presence of the test molecule. A decrease in the amount ofknown ligand bound to the γ subunit, a fragment thereof, or a fragmentcomprising the C1q, AdipoQ or ApM1 binding site thereof when the testmolecule is present indicates that the test molecule is able to bind tothe γ subunit, a fragment thereof, or a fragment comprising the C1q,AdipoQ or ApM1 binding site thereof. This method may be used to identifycompounds which bind to the γ subunit and which therefore representpotential agonists or antagonists of LSR activity which can be exploitedin the medicaments described above.

Proteins, peptides, drugs, small molecules, or other compoundsinteracting with the γ subunit, a fragment thereof, or a fragmentcomprising the C1q, AdipoQ or ApM1 binding site thereof can also bescreened by using an Optical Biosensor as described in Edwards etLeatherbarrow, Analytical Biochemistry, 246, 1-6 (1997), the disclosureof which is incorporated herein by reference. The main advantage of themethod is that it allows the determination of the association ratebetween the γ subunit and other interacting molecules. Thus, it ispossible to specifically select interacting molecules with a high or lowassociation rate. Typically a target molecule is linked to the sensorsurface (through a carboxymethyl dextran matrix) and a sample of testmolecules is placed in contact with the target molecules. The binding ofa test molecule to the target molecule causes a change in the refractiveindex and/or thickness. This change is detected by the Biosensorprovided it occurs in the evanescent field (which extend a few hundredmanometers from the sensor surface). In these screening assays, thetarget molecule can be the γ subunit, a fragment thereof, or a fragmentcomprising the C1q, AdipoQ or ApM1 binding site thereof and the testsample can be a collection of proteins extracted from tissues or cells,a pool of expressed proteins, combinatorial peptide and/or chemicallibraries, phage displayed peptides, drugs, small molecules or othercompounds. The tissues or cells from which the test proteins areextracted can originate from any species.

Proteins or other molecules interacting with the γ subunit, a fragmentthereof, or a fragment comprising the C1q, AdipoQ or ApM1 binding sitethereof can be also be found using affinity columns which contain the γsubunit, a fragment thereof, or a fragment comprising the C1q, AdipoQ orApM1 binding site thereof. The γ subunit, a fragment thereof, or afragment comprising the C1q, AdipoQ or ApM1 binding site thereof may beattached to the column using conventional techniques including chemicalcoupling to a suitable column matrix such as agarose, Affi Gel, or othermatrices familiar to those of skill in the art. In some versions of thismethod, the affinity column contains chimeric proteins in which the γsubunit, a fragment thereof, or a fragment comprising the C1q, AdipoQ orApM1 binding site thereof is fused to glutathione S-transferase. Amixture of cellular proteins or pool of expressed proteins as describedabove and is applied to the affinity column. Proteins, peptides, drugs,small molecules or other molecules interacting with the γ subunit, afragment thereof, or a fragment comprising the C1q, AdipoQ or ApM1binding site thereof attached to the column can then be isolated andanalyzed on 2-D electrophoresis gel as described in Ramunsen et al.Electrophoresis, 18, 588-598 (1997), the disclosure of which isincorporated herein by reference. Alternatively, the proteins or othermolecules retained on the affinity column can be purified byelectrophoresis based methods and sequenced. The same method can be usedto isolate antibodies, to screen phage display products, or to screenphage display human antibodies.

The compounds identified using the above methods may be screened todetermine whether they act as agonists or antagonists of LSR activity asfollows. Those compounds which are agonists will increase LSR activityin one or more assays selected from the group consisting of the rathepatocyte assay of Example 5, the postprandial lipemic response assayof Example 6, the plasma triglyceride assay of Example 7, the foodintake assay of Example 8, or the body weight assay of Example 7. Suchcompounds are useful in the medicaments discussed above for treatingconditions in which it is desirable to increase the partitioning ofdietary lipids to the liver.

Alternatively, those compounds which are antagonists of LSR activitywill inhibit the activity of AdipoQ in one or more assays selected fromthe group consisting of the rat hepatocyte assay of Example 5. thepostprandial lipemic response assay of Example 6, the plasmatriglyceride assay of Example 7, the food intake assay of Example 8, orthe body weight assay of Example 7. Such compounds are useful in themedicaments discussed above for treating conditions in which it isdesirable to reduce the partitioning of dietary lipids to the liver.

It will be appreciated that certain variations to this invention maysuggest themselves to those skilled in the art. The foregoing detaileddescription is to be clearly understood as given by way of illustration,the spirit and scope of this invention being interpreted upon referenceto the appended claims.

1. A method for treating obesity-related insulin resistance comprisingthe administration of a composition comprising an ApM1 polypeptidecomprising SEQ ID NO: 11 to an individual in an amount effective totreat the obesity-related insulin resistance.
 2. A method of treating anobesity-related condition selected from die group consisting ofobesity-related atherosclerosis, obesity-related hypertension,microangiopathic lesions resulting from abesity-related Type IIdiabetes, ocular lesions caused by microangiopathy in obese individualsit Type II diabetes, and renal lesions caused by microangiopathy inobese individuals with Typ II diabetes comprising the administration ofa composition comprising aphamaceutically acceptable carrier and an ApM1polypeptide comprising SEQ ID NO: 11 in an amount effective to treatsaid obesity-related condition.
 3. The method according to claim 2,wherein said obesity related condition is obesity-relatedatherosclerosis.
 4. The method according to claim 2, wherein saidobesity related condition is obesity-related hypertension.
 5. The methodaccording to claim 2, wherein said obesity related condition ismicroangiopathic lesions resulting from obesity-related Type IIdiabetes.
 6. The method according to claim 2, wherein said obesityrelated condition is ocular lesioning caused by microangiopathy in obeseindividuals with Type II diabetes.
 7. The method according to claim 2,wherein said obesity related condition is renal lesioning caused bymicroangiopathy in obese individuals with Type II diabetes.
 8. Themethod according to claim 3, wherein said composition is administered inan amount effective to treat obesity-related atherosclerosis.
 9. Themethod according to claim 4, wherein said composition is administered inan amount effective to treat obesity-related hypertension.
 10. Themethod according to claim 5, wherein said composition is administered inan amount effective to treat microangiopathic lesions resulting fromobesity-related Type II diabetes.
 11. The method according to claim 6,wherein said composition is administered in an amount affective to treatocular lesioning caused by microangiopathy in obese individuals withType II diabetes.
 12. The method according to claim 7, wherein saidcomposition is administered in an amount effective to treat renallesioning caused by microangiopathy in obese individuals with Type IIdiabetes.
 13. A method of increasing the partitioning of dietary lipidsto the liver comprising the administration of a composition comprising apharmaceutically acceptable carrier an ApM1 polypeptide comprising SEQID NO: 11 in an amount effective to increase the partitioning of dietarylipids to the liver.
 14. A method of reducing the levels of free fattyacids in obese individual comprising the administration of a compositioncomprising aphatmaceutically acceptable carrier and ApM1 polypeptidecomprising SEQ ID NO: 11 in an amount effective to reduce the levels offree fatty acids in obese individuals.